Cyclic dinucleotides as agonists of stimulator of interferon gene dependent signalling

ABSTRACT

Disclosed herein are new cyclic dinucleotide compounds and compositions and their application as pharmaceuticals for the treatment of disease. Methods of modulation of immune response to disease, and induce Stimulator of Interferon Genes (STING) dependent type I interferon production and co-regulated genes in a human or animal subject are also provided for the treatment diseases such as cancer, particularly metastatic solid tumors and lymphomas, inflammation, allergic and autoimmune disease, infectious disease, and for use as anti-viral agents and vaccine adjuvants.

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/461,642, filed Feb. 21, 2017, the contents of whichare incorporated by reference as if written herein in their entirety.

Disclosed herein are new cyclic dinucleotide compounds and compositionsand their application as pharmaceuticals for the treatment of disease,and methods of modulating immune response to disease, and induceStimulator of Interferon Genes (STING) dependent type I interferonproduction and co-regulated genes. Methods of modulation of STINGactivity in a human or animal subject are also provided for thetreatment of diseases such as cancer, particularly metastatic solidtumors and lymphomas, inflammation, allergic and autoimmune disease,infectious disease, and for use as vaccine adjuvants.

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defence to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and adaptive immunity. The innate immune system is the firstline of defence which is initiated by Pattern Recognition Receptors(PRRs) which detect ligands from the pathogens as well as damageassociated molecular patterns (Tokeuchi, O. et al., Cell 2010, 140,805-820). A growing number of these receptors have been identified,including Toll-like receptors (TLRs), C-type lectin receptors, retinoicacid inducible gene I (RIG-1)-like receptors and NOD-like receptors(NLRs) and also double stranded DNA sensors. Activation of PRRs leads toup-regulation of genes involved in the inflammatory response. Thisresponse utilizes such agents as: type 1 interferons, pro-inflammatorycytokines and chemokines, which suppress pathogen replication andfacilitate adaptive immunity.

The adaptor protein STING (Stimulator of Interferon Genes), also knownas TMEM 173, MPYS, MITA and ERIS, has been identified as a centralsignalling molecule in the innate immune response to cytosolic nucleicacids (Ishikawa, H.; Barber, G. N. Nature 2008, 455, 674-678;WO2013/1666000). Activation of STING results in up-regulation of IRFβand NFKB pathways leading to induction of interferon-β and othercytokines. STING is critical for responses to cytosolic DNA of pathogenor host origin, and of unusual nucleic acids called Cyclic Dinucleotides(CDNs). Initially characterized as ubiquitous bacterial secondarymessengers, CDNs [cyclic di-GMP (guanosine 5′-monophosphate) (CDG),cyclic di-AMP (adenosine 5′-monophosphate) (CDA), and cyclic GMP-AMP(cGAMP)] constitute a class of pathogen-associated molecular patternmolecules (PAMPs) that activate the TBK1/interferon regulatory factor 3(IRF3)/type 1 interferon (IFN) signaling axis via the cytoplasmicpattern recognition receptor stimulator of interferon genes (STING).Bacterial CDNs, such as CDG are symmetrical molecules characterised bytwo 3′, 5′ phophodiester linkages.

Direct activation of STING by bacterial CDNs has recently been confirmedthrough X-ray crystallography (Burdette, D. L.; Vance, R. E. NatureImmunology 2013, 14, 19-26; Cai, X. et al. Molecular Cell 2014, 54,289-296). More recently, the response to cytosolic DNA has beenelucidated and shown to involve generation, by an enzyme called cyclicGMP-AMP synthase (cGAS, previously known as C6orf150 or MB21D1), of anovel mammalian CDN signalling molecule identified as cGAMP, which thenactivates STING. Unlike bacterial CDNs, cGAMP is an unsymmetricalmolecule characterised by its mixed 2′,5′ and 3′,5′ phosphodiesterlinkages. (Gao, P. et al. Cell 2013, 153, 1-14).

Interferon was first described as a substance which could protect cellsfrom viral infection (Isaacs, A.; Lindemann, J. Proc. Royal Soc. Lon.Ser. B. Biol. Sci. 1957, 147, 258-267). Interferons belong to the classof small proteins known as cytokines, which are implicated inintercellular signalling. In humans, the type I interferons are a familyof related proteins encoded by genes on chromosome 9 and encoding atleast 13 isoforms of interferon alpha (IFNα) and one isoform ofinterferon beta (IFNβ). Recombinant IFNα was the first approvedbiological therapeutic and has become an important therapy in viralinfections and in cancer. As well as direct antiviral activity on cells,interferons are known to be potent agonists of the immune response,acting on cells of the immune system.

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines, could become an important strategy for thetreatment or prevention of human diseases including viral infections.Compounds capable of stimulating the immune response may be useful notonly in infectious diseases but also in cancer (Krieg, Curr. Oncol. Rep.2004, 6(2), 88-95), allergic diseases (Moisan J. et al. Am. J Physiol.Lung CellMol. Physiol. 2006, 290, 1987-1995), other inflammatoryconditions such as irritable bowel disease (Rakoff-Nahoum, S. et al.Cell 2004, 23, 118(2), 229-241), and as vaccine adjuvants (Persing, D.H. et al. Trends Microbiol. 2002, 10(10 Suppl), S32-S37).

Induction of type 1 interferons by activation of STING, may offerbenefit in treatment of allergic diseases such as asthma and allergicrhinitis (Huber, J. P. et al. J. Immunol. 2010, 185, 813-817). Recentevidence has been accumulated to suggest that allergen-reactive type 2helper T cells (Th2) play a triggering role in the activation and/orrecruitment of IgE antibody-producing B cells, mast cells andeosinophils, all of which are involved in the allergic inflammation. Th2responses are associated with raised levels of IgE, which, via itseffects on mast cells, promotes a hypersensitivity to allergens,resulting in the symptoms seen, for example, in allergic rhinitis andasthma. In healthy individuals the immune-response to allergens is morebalanced with a mixed Th2/Th1 and regulatory T cell response. Inductionof type 1 interferons have been shown to result in reduction of Th2-typecytokines in the local environment and promote Th1/Treg responses.

Activation of the stimulator of interferon genes (STING) pathway hasbeen identified as one of the key determinants in the generation of thespontaneous T-cell response in vivo. The STING pathway is activated bytumor-derived DNA in the cytosol of dendritic cells (DCs), which in turnresults in increased levels of cGAMP. Antigen-presenting cells (APCs)such as DCs are implicated in the antitumor T cell response pathway.Upon exposure to immune danger signals, DCs act as potent T cellstimulators, via induction of cGAS, followed by production of cGAMP,which in turn binds to STING. This pathway induces the phenotypicmaturation of DCs, followed by the production of type I interferon andother cytokines. Activation of STING induces transcription of numeroushost defence genes, resulting in the production of interferon beta,chemokine release and ultimately priming of antigen-specific T-cells,thus eliciting spontaneous anti-tumor T cell response. Innate immuneactivation by STING agonists in the tumor microenvironment leads tovascular disruption and rapid tumor collapse, which is followed by Tcell-mediated adaptive immunity against residual disease.Pharmacological stimulation of the STING pathway with chemicallymodified cyclic dinucleotides has proven therapeutically efficaciouspreclinically against tumors in relevant in vivo models, and offersgreat promise in the clinical setting, with the first STING agonistscurrently in Phase I studies in patients with metastatic solid tumors orlymphomas. Intratumoral injection of an optimized STING agonist iscurrently being tested clinically in a phase I clinical trial inadvanced cancer patients (NCT02675439).

Induction of IFN in Listeria-infected cells is STING-dependent, and itis lost in goldenticket mice harboring a mutant STING allele. Otherobservations, including the CDG-STING cocrystal structures, demonstratedthat cytosolic microbial CDNs bind to STING to elicit an IFN andproinflammatory signaling cascade. The STING signaling pathway hasemerged as a central TLR-independent mediator of host innate defensestimulated by cytosolic nucleic acids, either through direct binding ofexogenous CDNs from bacteria or through binding of a structurallydistinct CDN produced by a host cGAS in response to cytosolicdouble-stranded DNA (dsDNA). The STING pathway represents a central nodelinking cytosolic nucleic acids to a transcriptional response resultingin a MyD88-independent production of type I IFN.

Modulation of STING holds promise for the development of compounds withantiviral activity. Recognition of pathogens is mainly mediated by PRRs,including TLRs, RIG-I-like receptors (RLRs) and NOD-like receptors(NLRs) (Takeuchi, O. et al. Cell 2010, 140, 805-820), that triggersignal cascades to upregulate the expression of various cytokines. Inthe case of viral infection, endosomal TLRs and cytoplasmic RLRs detectviral DNAs or RNAs and induce the production of type I IFN, which arepotent inhibitors of viral replication (Gitlin, L. et al. PNAS USA 2006,103, 8459-8464; Kato, H. et al. Nature Immunol. 2005, 23, 19-28; Kato,H. et al. Nature 2006, 441, 101-105). STING has been shown to activatedownstream transcription factors STAT6 and IRF3 through TBK1, which areresponsible for antiviral response and innate immune response againstintracellular pathogen. It has been recently reported that, in responseto viral infection, STING activates STAT6 (signal transducer andactivator of transcription 6) to induce (Th2-type), increase (IL-12) ordecrease (IL-10) production of various cytokines, including thechemokines CCL2, CCL20, and CCL26 (Chen, H. et al. Cell 2011, 147,436-446).

Compounds and pharmaceutical compositions, certain of which have beenfound to modulate STING activity have been discovered, together withmethods of synthesizing and using the compounds including methods forthe treatment of STING-mediated diseases in a patient by administeringthe compounds.

The present invention also relates to new adjuvants and their uses inpharmaceutical compositions, such as vaccines. In particular, thepresent invention provides new compounds useful as adjuvants and/orimmunomodulators for prophylactic and/or therapeutic vaccination in thetreatment of infectious diseases, inflammatory diseases, autoimmunediseases, tumours, allergies as well as for the control of fertility inhuman or animal populations.

Vaccination has become the most cost-effective measure to preventinfections. However, there are still many diseases for which vaccinesare not yet available or the available vaccines are not completelysatisfactory due to low efficacy, high reactogenicity, poor stabilityand/or high costs. Thus, there is still an urgent need for both new andimproved vaccines.

Recent findings suggest that vaccines can also be used in theimmunotherapy of transmissible diseases. Furthermore, vaccines can beused in prophylaxis or immunotherapy of autoimmune diseases,inflammatory diseases, tumours, allergies and for the control offertility in human and/or animal populations.

The use of optimal adjuvants plays a crucial role in vaccination.Antigens administered without adjuvant only rarely mediate an adequateimmune response. In addition, not only the strength but also the qualityof the elicited immune response matters. Stimulation of an incorrectimmunization pattern may lead to immunopathological reactions andexacerbation of the symptoms of infection. In this context, the adjuvantcan help to assist the desired immune response. In other words, anadjuvant can modulate the immune response or redirect the immuneresponse to balance the immune response in the desired direction.

Substances referred to as “adjuvants” are those which are added and/orco-formulated in an immunization to the actual antigen (i.e. thesubstance which provokes the desired immune response) in order toenhance the humoral and/or cell-mediated immune response (“Lexikon derBiochemie und Molekularbiologie”, 1. Band, Spektrum, Akademischer Verlag1995). That is, adjuvants are compounds having immunopotentiatingproperties, in particular, when co-administered with antigens. The useof many adjuvants is based solely on experience, and the effect canneither be accurately explained nor predicted. The following groups ofadjuvants are traditionally used in particular: aluminum hydroxide,emulsions of mineral oils, saponins, detergents, silicon compounds,thiourea, endotoxins of gram-negative bacteria, exotoxins ofgram-positive bacteria, killed or attenuated living bacteria or partsthereof.

As adjuvants which may be useful in mucosal vaccination the followinghave been described: The MALP-2 molecule andbisacyloxypropylcysteine-conjugates thereof, e.g. abispalmitoyloxypropylcysteine-PEG molecule is known to represent potentstimulants for macrophages. The usefulness of MALP-2 as an adjuvant wasshown previously, see e.g. WO2004/009125 and WO2003/084568. Inparticular, it was demonstrated that MALP-2 can act as an effectivemucosal adjuvant enhancing the mucosal immune response, e.g. fosteringan enhanced expression of antigen-specific IgA antibodies.

Furthermore, it was shown that MALP-2 can activate dendritic cells andB-cells, both play an important role in the induction of a specifichumoral immune response. In addition, preliminary studies demonstratethat a combination of biologically active HIV-1 tat protein andsynthetic MALP-2 may be a promising vaccine with the MALP-2 component asan effective mucosal adjuvant.

There has been an intensive search in recent years for novel adjuvants,including those for the mucosal administration route. Only a fewsubstances have been found to be able to enhance mucosal responses.Among these, some act as carriers to which the antigens must be bound orfused thereto. Far fewer universally employable “true” adjuvants whichare admixed to the antigens have been found, as outlined above.

Prokaryotic as well as eukaryotic cells use various small molecules forcell signaling and intra- and intercellular communication. For example,cyclic nucleotides like cGMP, cAMP, etc. are known to have regulatoryand initiating activity in pro- and eukaryotic cells. While ineukaryotic cells cAMP and cGMP are used as signalling molecules,prokaryotic cells utilize cyclic di-nucleoside mono phosphate molecules,in particular CDG, beside cAMP.

The condensation of two GTP molecules is catalyzed by the enzymediguanylate cyclase (DGC) to give CDG, which has demonstratedanti-microbial activity, and which may be used to prevent or combatpathogens. Moreover, CDG acts as a key bacterial regulator: in bacterialcells, CDG regulates the expression of genes and the biosynthesis ofexo-polysaccharides. Further, CDG has not been implicated in anyeukaryotic biochemical pathway. Since interacting ligands of CDG areexpressed throughout the various genera of bacteria, it is assumed thatmost bacteria use CDG as a regulatory molecule.

Bacterial CDNs and their analogues have consequently attracted interestas potential vaccine adjuvants (Libanova R. et al. MicrobialBiotechnology 2012, 5, 168-176; WO2007/054279, WO2005/087238). In WO2005/087238, it has been speculated that CDG or analogs thereof canstimulate or enhance immune or inflammatory response in a patient or canenhance the immune response to a vaccine by serving as an adjuvant.Further, it is speculated that CDG or its analogs may be used as activeingredient in compositions for treating injuries, diseases, disordersand conditions that result in neurodegeneration. Therein, data areprovided showing that cyclic diGMP does not modulate DC endocyticactivity but may activate dendritic cells due to induction of expressionof co-stimulatory molecules. Further, data are provided showing thatoccasionally CDG may upregulate immunostimulatory capacity of dendriticcells. Further, data are provided showing that CDG in high doses mayactivate T-cells in vitro when mixed with dendritic cells. However, anyenhancement of immune or inflammatory responses in a patient orenhancement of the immune response to a vaccine by serving as anadjuvant is not shown, rather it is speculated therein that there aresome data which may indicate for an increased presentation of antigenthrough stimulation of HLA-DR. Further, no immunomodulatory action ofcyclic diGMP is shown in said document. Hence, this document merelyspeculates about any immunomodulatory, in particular, about any enhancedimmune response by serving as an adjuvant. As discussed before, anadjuvant is a compound able to provoke or enhance the humoral and/orcell mediated immune response against an active antigen. No data areprovided in WO 2005/087238 showing an immune response against an activeantigen using CDG as adjuvant for enhancing or eliciting or modulatingsaid immune response. In addition, it is noted that said document onlyprovides information regarding CDG but not with respect to any otheranalogs of cyclic diGMP.

The discovery of the STING pathway, and CDNs that activate it opened upseveral new possibilities for the development of vaccine adjuvants.STING agonists would be candidates for clinical testing as adjuvants andas stimulants for anticancer immune activity. DMXAA, which markedlyshrinks cancer in mouse model systems by activating the innate immuneresponse, is a STING agonist that activates mouse STING (mSTING) but nothSTING. To test the therapeutic hypothesis that STING agonists will beeffective for cancer treatment or as vaccine adjuvants, molecules thatare active in humans are required. Analogues to cGAMP that are resistantto its activity have been tested for use as vaccine adjuvants (Li, L. etal. Nature Chem. Biol. 2014, 10, 1043-1048).

There is a need to provide new compounds useful as adjuvants for usewith vaccines. In particular, there is a need for adjuvants which canelicit a strong immune response which represent a balanced or adjustedimmune response involving both humoral and cellular components, thus,allowing effective prophylaxis or treatment of various diseases andconditions, specifically of infectious diseases or cancer.

Thus, an object of the present disclosure is the provision of adjuvantswhich can elicit, enhance, or modulate (pre-existing) immune response inan individual or subject. In particular, the disclosure is directedtowards development of a range of novel, highly active adjuvants,particularly, but not limited to, mucosal adjuvants which are non-toxicfor humans and which can be employed with a wide variety of activeingredients to be assisted in conventional or novel vaccines such as, inparticular, prophylactic or therapeutic vaccines, including cancer andDNA vaccines.

In certain embodiments of the present invention, compounds havestructural Formula Ia:

or a salt, ester, tautomer, or prodrug thereof, wherein:

A₁ and A₂ are independently selected from CH and N;

R_(1a) and R_(1b) are independently selected from H and NH₂;

R₂ is selected from OH, F, Cl, N₃, and NH₂;

R₃ is selected from OH, F, Cl, N₃, and NH₂;

R_(4a) and R_(4b) are independently selected from NH₂, OH, NHR₅, andOR₅; and

R₅ is independently selected from methyl, ethyl, and propyl; with theprovisos that:

when A₁ is CH, then R_(4a) is not NHR₅ or OR₅;

when A₂ is CH, then R_(4b) is not NHR₅ or OR₅; and

when A₁ and A₂ are both N, then at least one of R₂ and R₃ is not OH.

Also provided are all stereoisomers, including enantiomers anddiastereomers, of compounds of Formula Ia.

In certain embodiments of the present invention, compounds havestructural Formula

or a salt, ester, tautomer, or prodrug thereof, wherein:

A₁ and A₂ are independently selected from CH and N;

R_(1a) and R_(1b) are independently selected from H and NH₂;

R₂ is selected from OH, F, Cl, N₃, and NH₂;

R₃ is selected from OH, F, Cl, N₃, and NH₂;

R_(4a) and R_(4b) are independently selected from NH₂, OH, NHR₅, andOR₅; and

R₅ is independently selected from methyl, ethyl, and propyl; with theprovisos that:

when A₁ is CH, then R_(4a) is not NHR₅ or OR₅;

when A₂ is CH, then R_(4b) is not NHR₅ or OR₅; and

when A₁ and A₂ are both N, then at least one of R₂ and R₃ is not OH.

Certain compounds disclosed herein may possess useful STING modulatingactivity, and may be used in the treatment or prophylaxis of a diseaseor condition in which STING plays an active role. Thus, in broad aspect,certain embodiments also provide pharmaceutical compositions comprisingone or more compounds disclosed herein together with a pharmaceuticallyacceptable carrier, as well as methods of making and using the compoundsand compositions. Certain embodiments provide methods for modulatingSTING. Other embodiments provide methods for treating a STING-mediateddisorder in a patient in need of such treatment, comprisingadministering to said patient a therapeutically effective amount of acompound or composition according to the present invention. Alsoprovided is the use of certain compounds disclosed herein for use in themanufacture of a medicament for the treatment of a disease or conditionameliorated by the agonism of STING.

It will be appreciated by a person in the chemical arts that compoundsof Formula (Ia) possess several asymmetric, tetrahedral atoms. Formula(Ia) embraces compounds that possess all possible combinations ofabsolute stereochemistry at the various asymmetric, tetrahedral atoms.Due to the nonidentical substitution at the two tetrahedral phosphorusatoms, each atom represents a center of chirality. Formula (Ia) embracescompounds that possess all possible combinations of absolutestereochemistry at the two asymmetric, tetrahedral phosphorus atoms.

In certain embodiments of the present invention, compounds havestructural Formula IIa:

or a salt, ester, tautomer, or prodrug thereof, wherein:

A₁ and A₂ are independently selected from CH and N;

R_(1a) and R_(1b) are independently selected from H and NH₂;

R₂ is selected from OH, F, and Cl;

R₃ is OH;

R_(4a) and R_(4b) are independently selected from NH₂ and OH; and

when A₁ and A₂ are both N, then R₂ is not OH.

Also provided are stereoisomeric forms of Formula IIa, includingenantiomers and diastereomers for which the phosphorus indicated inFormula IIa has R absolute stereochemistry.

In certain embodiments of the present invention, compounds havestructural Formula II:

or a salt, ester, tautomer, or prodrug thereof, wherein:

R₄ and A₂ are independently selected from CH and N;

R_(1a) and R_(1b) are independently selected from H and NH₂;

R₂ is selected from OH, F, and Cl;

R₃ is OH;

R_(4a) and R_(4b) are independently selected from NH₂ and OH; and

when A₁ and A₂ are both N, then R₂ is not OH.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, A₁ is CH andA₂ is N.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, A₁ is N andA₂ is CH.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, A₁ and A₂are both CH.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, A₁ and A₂are both N.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, A₁ and A₂are both N, and R₂ is selected from F and Cl.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, R_(1a) andR_(1b) are both H.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, R_(4a) andR_(4b) are independently selected from NH₂ and NHR₅.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, R_(4a) andR_(4b) are independently selected from NH₂ and NHR₅, and R_(1a) andR_(1b) are both H.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, R_(4a) andR_(4b) are both NH₂.

In certain embodiments of any of Formulas I, Ia, 2, and 2a, R_(4a) andR_(4b) are both NH₂, and R_(1a) and R_(1b) are both H.

Also provided is a compound chosen from the species described herein,e.g.:

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from:

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from:

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from:

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from

or a salt, ester, tautomer, or prodrug thereof.

In certain embodiments, the compound is selected from

or a salt, ester, tautomer, or prodrug thereof.

Also provided is a compound chosen from the Examples disclosed herein,or a salt, ester, tautomer, or prodrug thereof.

Also provided are embodiments wherein a compound of Formula Ia isconjugated to a targeting moiety for targeted delivery.

In certain embodiments, the targeting moiety is a biotin or biotinanalogue.

In certain embodiments, the targeting moiety is a peptide.

In certain embodiments, the targeting moiety is a protein.

In certain embodiments, the protein is transferrin.

In certain embodiments, the targeting moiety is an antibody.

Also provided are embodiments wherein a compound of Formula Ia isconjugated to an antibody for targeted delivery.

In certain embodiments, the antibody is a monoclonal antibody.

Also provided are embodiments wherein a compound of Formula Ia isconjugated to a hapten for binding to an antibody for targeted delivery.

Also provided are embodiments wherein a compound of Formula Ia isconjugated to a nanoparticle for targeted delivery.

In certain embodiments, the nanoparticle is comprised of polymers of oneor more alpha-hydroxycarboxylic acids.

Also provided are embodiments wherein a compound of Formula Ia iscontained within a polymeric delivery vehicle.

Also provided are embodiments wherein a compound of Formula Ia iscontained within a liposome.

Also provided are embodiments wherein a compound of Formula Ia iscontained within a micelle.

Also provided are embodiments wherein a compound of Formula Ia iscontained within a vesicle.

In certain embodiments, the vesicle is comprised of phospholipids.

Also provided are embodiments wherein any embodiment above may becombined with any one or more of these embodiments, provided thecombination is not mutually exclusive.

As used herein, two embodiments are “mutually exclusive” when one isdefined to be something which is different than the other. For example,an embodiment wherein two groups combine to form a cycloalkyl ismutually exclusive with an embodiment in which one group is ethyl theother group is hydrogen. Similarly, an embodiment wherein one group isCH₂ is mutually exclusive with an embodiment wherein the same group isNH.

The present invention also relates to a method of inhibiting at leastone STING function comprising the step of contacting STING with acompound as described herein. The cell phenotype, cell proliferation,activity of STING, change in biochemical output produced by activeSTING, expression of STING, or binding of STING with a natural bindingpartner may be monitored. Such methods may be modes of treatment ofdisease, biological assays, cellular assays, biochemical assays, or thelike.

Also provided herein is a method of treatment of a STING-mediateddisease comprising the administration of a therapeutically effectiveamount of a compound as disclosed herein, or a salt thereof, to apatient in need thereof.

In certain embodiments, the STING-mediated disease is an autoimmunedisease or disorder.

In certain embodiments, the STING-mediated disease is an immunedeficiency or defect.

In certain embodiments, the STING-mediated disease is an inflammatorydisease or disorder.

In certain embodiments, the STING-mediated disease is cancer.

In certain embodiments, the cancer is chosen from a metastatic solidtumor and lymphoma. Also provided herein is a compound as disclosedherein for use as a medicament.

Also provided herein is a compound as disclosed herein for use as amedicament for the treatment of a STING-mediated disease.

Also provided is the use of a compound as disclosed herein as amedicament.

Also provided is the use of a compound as disclosed herein as amedicament for the treatment of a STING-mediated disease.

Also provided is a compound as disclosed herein for use in themanufacture of a medicament for the treatment of a STING-mediateddisease.

Also provided is the use of a compound as disclosed herein for thetreatment of a STING-mediated disease.

Also provided herein is a method of agonizing STING comprisingcontacting STING with a compound as disclosed herein, or a salt thereof.

Also provided herein is a method for achieving an effect in a patientcomprising the administration of a therapeutically effective amount of acompound as disclosed herein, or a salt thereof, to a patient, whereinthe effect is chosen from induction of transcription of host defensegenes, production of interferon beta, release of chemokines, priming ofantigen-specific T-cells.

In certain embodiments, the STING-mediated disease is an autoimmunedisease or disorder.

In certain embodiments, the STING-mediated disease is an immunedeficiency or defect.

In certain embodiments, the STING-mediated disease is an inflammatorydisease.

In certain embodiments, the STING-mediated disease is cancer.

In certain embodiments, the cancer is chosen from a metastatic solidtumor and lymphoma.

Also provided is a method of modulation of a STING-mediated function ina subject comprising the administration of a therapeutically effectiveamount of a compound as disclosed herein.

Also provided is a pharmaceutical composition comprising a compound asdisclosed herein, together with a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition is formulated fororal administration.

In certain embodiments, the oral pharmaceutical composition is chosenfrom a tablet and a capsule.

In certain embodiments, the pharmaceutical composition is formulated forparenteral administration. In certain embodiments, the parenteraladministration is chosen from subcutaneous, intravenous, intramuscular,intraaterial, intraderminal, intrathecal, and epidural.

In certain embodiments, the pharmaceutical composition is formulated forintratumoral administration.

Terms

As used herein, the terms below have the meanings indicated.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n₂ are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycle, or any other moiety were the atom attached to the carbonylis carbon. An “acetyl” group refers to a —C(O)CH₃ group. An“alkylcarbonyl” or “alkanoyl” group refers to an alkyl group attached tothe parent molecular moiety through a carbonyl group. Examples of suchgroups include methylcarbonyl and ethylcarbonyl. Examples of acyl groupsinclude formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—), (—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, said alkyl will comprise from 1 to10 carbon atoms. In further embodiments, said alkyl will comprise from 1to 8 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, noyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH₂—). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R—S—) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, said alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:::C—, —C≡C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl,” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(O)N(RR′) group with R and R′ as defined herein or as defined by thespecifically enumerated “R” groups designated. The term “N-amido” asused herein, alone or in combination, refers to a RC(O)N(R′)— group,with R and R′ as defined herein or as defined by the specificallyenumerated “R” groups designated. The term “acylamino” as used herein,alone or in combination, embraces an acyl group attached to the parentmoiety through an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR, wherein R and R′ are independently chosen from hydrogen, alkyl,acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl,any of which may themselves be optionally substituted. Additionally, Rand R′ may combine to form heterocycloalkyl, either of which may beoptionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch polycyclic ring systems are fused together. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl,phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C₆H₄=derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group-with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′— group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxy,” as used herein, refers to —C(O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt. An “O-carboxy” group refers to a RC(O)O— group, where R is asdefined herein. A “C-carboxy” group refers to a —C(O)OR groups where Ris as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl group wherein each cyclic moietycontains from 3 to 12 carbon atom ring members and which may optionallybe a benzo fused ring system which is optionally substituted as definedherein. In certain embodiments, said cycloalkyl will comprise from 5 to7 carbon atoms. Examples of such cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronapthyl,indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and thelike. “Bicyclic” and “tricyclic” as used herein are intended to includeboth fused ring systems, such as decahydronaphthalene,octahydronaphthalene as well as the multicyclic (multicentered)saturated or partially unsaturated type. The latter type of isomer isexemplified in general by, bicyclo[1.1.1]pentane, camphor, adamantane,and bicyclo[3.2.1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or combinations thereof, fullysaturated or containing from 1 to 3 degrees of unsaturation, consistingof the stated number of carbon atoms and from one to three heteroatomschosen from N, O, and S, and wherein the N and S atoms may optionally beoxidized and the N heteroatom may optionally be quaternized. Theheteroatom(s) may be placed at any interior position of the heteroalkylgroup. Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃.

The term “heteroaryl,” as used herein, alone or in combination, refersto a 3 to 15 membered unsaturated heteromonocyclic ring, or a fusedmonocyclic, bicyclic, or tricyclic ring system in which at least one ofthe fused rings is aromatic, which contains at least one atom chosenfrom N, O, and S. In certain embodiments, said heteroaryl will comprisefrom 1 to 4 heteroatoms as ring members. In further embodiments, saidheteroaryl will comprise from 1 to 2 heteroatoms as ring members. Incertain embodiments, said heteroaryl will comprise from 5 to 7 atoms.The term also embraces fused polycyclic groups wherein heterocyclicrings are fused with aryl rings, wherein heteroaryl rings are fused withother heteroaryl rings, wherein heteroaryl rings are fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated (but nonaromatic)monocyclic, bicyclic, or tricyclic heterocyclic group containing atleast one heteroatom as a ring member, wherein each said heteroatom maybe independently chosen from nitrogen, oxygen, and sulfur. In certainembodiments, said hetercycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, said hetercycloalkyl willcomprise from 1 to 2 heteroatoms as ring members. In certainembodiments, said hetercycloalkyl will comprise from 3 to 8 ring membersin each ring. In further embodiments, said hetercycloalkyl will comprisefrom 3 to 7 ring members in each ring. In yet further embodiments, saidhetercycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Examples ofheterocycle groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl,dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl,dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl,benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl,1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl,pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and thelike. The heterocycle groups may be optionally substituted unlessspecifically prohibited.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The phrase “in the main chain” refers to the longest contiguous oradjacent chain of carbon atoms starting at the point of attachment of agroup to the compounds of any one of the formulas disclosed herein.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms (i.e., C₁-C₆ alkyl).

The term “lower aryl,” as used herein, alone or in combination, meansphenyl or naphthyl, either of which may be optionally substituted asprovided.

The term “lower heteroaryl,” as used herein, alone or in combination,means either 1) monocyclic heteroaryl comprising five or six ringmembers, of which between one and four said members may be heteroatomschosen from N, O, and S, or 2) bicyclic heteroaryl, wherein each of thefused rings comprises five or six ring members, comprising between themone to four heteroatoms chosen from N, O, and S.

The term “lower cycloalkyl,” as used herein, alone or in combination,means a monocyclic cycloalkyl having between three and six ring members(i.e., C₃-C₆ cycloalkyl). Lower cycloalkyls may be unsaturated. Examplesof lower cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

The term “lower heterocycloalkyl,” as used herein, alone or incombination, means a monocyclic heterocycloalkyl having between threeand six ring members, of which between one and four may be heteroatomschosen from N, O, and S (i.e., C₃-C₆ heterocycloalkyl). Examples oflower heterocycloalkyls include pyrrolidinyl, imidazolidinyl,pyrazolidinyl, piperidinyl, piperazinyl, and morpholinyl. Lowerheterocycloalkyls may be unsaturated.

The term “lower amino,” as used herein, alone or in combination, refersto —NRR′, wherein R and R′ are independently chosen from hydrogen andlower alkyl, either of which may be optionally substituted.

The term “mercaptyl” as used herein, alone or in combination, refers toan RS— group, where R is as defined herein.

The term “mercapto” as used herein, alone or in combination, refers toan —SH group.

The term “phosphate”, as used herein, alone or in combination, refers tothe functional group P(═O)(OR_(X))(OR_(Y))(OR_(Z)), wherein R_(X),R_(Y), and R_(Z) are independently chosen from hydrogen and organicgroups. The term can also refer to a compound containing this functionalgroup.

The term “phosphodiester”, as used herein, alone in combination, refersto the functional group P(═X)(OR_(X))(OR_(Y))(OH), wherein R_(X) andR_(Y) are organic groups, and X is selected from oxygen and sulfur.

The term “thiophosphate”, as used herein, alone or in combination,refers to a phosphate functional group in which one or more of theoxygen atoms has been replaced with sulfur. The term can also refer to acompound containing this functional group.

The term “thiolophosphate”, as used herein, alone or in combination,refers to the phosphate functional group which contains a P—S—R moietyin place of a P—O—R moiety. This term can also refer to a compoundcontaining this functional group.

The term “thionophosphate”, as used herein, alone or in combination,refers to a phosphate functional group in which the P═O moiety has beenreplaced with a P═S moiety. The term can also refer to a compoundcontaining this functional group.

It will be understood that, for certain thiolophosphates andthionophosphates, the following tautomeric equilibrium can occur:P(═S)(OR_(X))(OR_(Y))(OH)←→P(═O)(OR_(X))(OR_(Y))(SH)

The term “thiono” as used herein, alone or in combination, refers to ═S.

The term “nitro,” as used herein, alone or in combination, refers to—NO₂.

The terms “oxy” or “oxa,” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refer the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “tautomer”, as use herein, alone or in combination, refers toone of two or more isomers that rapidly interconvert. Generally, thisinterconversion is sufficiently fast so that an individual tautomer isnot isolated in the absence of another tautomer. The ratio of the amountof tautomers can be dependent on solvent composition, ionic strength,and pH, as well as other solution parameters. The ratio of the amount oftautomers can be different in a particular solution and in themicroenvironment of a biomolecular binding site in said solution.Examples of tautomers that are well known in the art include keto/enol,enamine/imine, and lactam/lactim tautomers. Examples of tautomers thatare well known in the art also include 2-hydroxypyridine/2(1H)-pyridoneand 2-aminopyridine/2(1H)-iminopyridone tautomers.

The term “predominantly one isomer”, as used herein, means that acompound contains at least about 85% of one isomer (e.g., an enantiomeror diastereomer). For example, in certain embodiments, a compound maycontain at least about 90% of one isomer. In certain embodiments, acompound may contain at least about 95% of one isomer. In certainembodiments, a compound may contain at least about 98% of one isomer. Incertain embodiments, a compound may contain at least about 99% of oneisomer. Similarly, the phrase “substantially free from other isomers”means that the compound contains at most about 15% of another isomer.For example, in certain embodiments, the compound contains at most about10% of another isomer. In certain embodiments, the compound contains atmost about 5% of another isomer. In certain embodiments, the compoundcontains at most about 2% of another isomer. In certain embodiments, thecompound contains at most about 1% of another isomer.

Cyclic dinucleotides, including those specifically described herein, aswell as isoforms (e.g., tautomers) of those specifically describedherein that can be used in practicing the invention. Cyclicdinucleotides can be obtained using any suitable method. For example,cyclic dinucleotides may be made by chemical synthesis using nucleosidederivatives as starting material. Cyclic dinucleotides can also beproduced by in vitro synthesis, using recombinant purified cGAMPsynthase. Moreover, the structures of such cyclic dinucleotides can beconfirmed using analytical chemical techniques. These techniquesinclude, but are not limited to, nuclear magnetic resonance (proton andother nuclei, and both 1D and 2D), X-ray crystallography,electromagnetic spectroscopy (including but not limited to visible,infrared, optical regions of the electromagnetic spectrum, and includingbut not limited to absorption, emission, and optical rotation methods)and mass spectrometry.

Cyclic dinucleotides provided herein can be described by the followingnomenclature: cyclic[X₁(A-5′)pX₂(B-5′)p], wherein X₁ and X₂ are thefirst and second nucleosides, A is the point of attachment of the firstnucleoside (e.g. 2′ or 3′) that is linked to the 5′ carbon of the secondnucleoside via a phosphodiester bond, and B is the point of attachmentof the second nucleoside (e.g. 2′ or 3′) that is linked to the 5′ carbonof the first nucleoside. For instance, based on this nomenclature,cyclic(A(2′-5′)pG(3′-5′)p] has the following formula:

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “sulfhydryl,” as used herein, alone or in combination, refersto an —SH group.

The term “thiono”, as used herein, alone or in combination, refers to asulfur doubly bonded to the parent atom.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocarbamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that said groupis absent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Wherestructurally feasible, two substituents may be joined together to form afused five-, six-, or seven-membered carbocyclic or heterocyclic ringconsisting of zero to three heteroatoms, for example formingmethylenedioxy or ethylenedioxy. An optionally substituted group may beunsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃),monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywherein-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Wheresubstituents are recited without qualification as to substitution, bothsubstituted and unsubstituted forms are encompassed. Where a substituentis qualified as “substituted,” the substituted form is specificallyintended. Additionally, different sets of optional substituents to aparticular moiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety chosen fromhydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl andheterocycloalkyl, any of which may be optionally substituted. Such R andR′ groups should be understood to be optionally substituted as definedherein. Whether an R group has a number designation or not, every Rgroup, including R, R′ and R^(n) where n=(1, 2, 3, . . . n), everysubstituent, and every term should be understood to be independent ofevery other in terms of selection from a group. Should any variable,substituent, or term (e.g. aryl, heterocycle, R, etc.) occur more thanone time in a formula or generic structure, its definition at eachoccurrence is independent of the definition at every other occurrence.Those of skill in the art will further recognize that certain groups maybe attached to a parent molecule or may occupy a position in a chain ofelements from either end as written. For example, an unsymmetrical groupsuch as —C(O)N(R)— may be attached to the parent moiety at either thecarbon or the nitrogen.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral atom. It will beunderstood by a person in the art that any tetrahedral atom with fornonidentical substituents can possess a chiral center. The group of suchatoms includes, but is not limited to, carbon and phosphorus. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and l-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentinvention includes all cis, trans, syn, anti, entgegen (E), and zusammen(Z) isomers as well as the appropriate mixtures thereof. Additionally,compounds may exist as tautomers; all tautomeric isomers are provided bythis invention. Additionally, the compounds disclosed herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms.

The terms “R” and “S” will be understood by persons of skill in thechemical art to refer to the absolute stereochemistry at a givenasymmetric tetrahedral atom, using the Cahn-Ingold-Prelog rules forpriority. A chemical structure provided herein having an asymmetrictetrahedral atom will embrace compounds having either “R” or “S”absolute stereochemistry at said atom, in the absence of either (a) aclear “R” or “S” designation in the structure at said atom, or (b) clearwedge and dash depictions of bonds to said atom, as understood bypersons of skill in the chemical art to indicate absolutestereochemistry.

The terms “R_(P)” and “S_(P)” refer to absolute stereochemistry at aphosphorus atom. For compounds with more than one phosphorus atom, andthus requiring more than one such term, the terms will be listed in thesame order as is given for the phosphorus atoms in the name provided.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

As used herein, alone or in combination, the term “supramolecularassembly” refers to an assembly of molecules held together bynoncovalent interactions. In some embodiments, the nocovalentinteraction is a hydrophobic interaction. In some embodiments, thenoncovalent interaction is a hydrogen bonding interaction. In someembodiments, the noncovalent interaction is electrostatic interactionbetween ions. In some embodiments, the molecules are amphiphilic innature. In some embodiments, the supramolecular assemblies comprise bothhydrophilic and hydrophobic regions. In some embodiments, thesupramolecular assemblies comprise lipid bilayers.

As used herein, alone or in combination, the term “nanoparticle” refersto a particle larger than conventional synthetic organic molecules. Insome embodiments, nanoparticles are greater than about 100 picometers indiameter. In some embodiments, nanoparticles are greater than about 1nanometer in diameter. In some embodiments, nanoparticles are greaterthan about 10 nanometers in diameter. In some embodiments, nanoparticlesare greater than about 100 nanometers in diameter. In some embodiments,nanoparticles are greater than about 1 micrometer in diameter. In someembodiments, nanoparticles are greater than about 10 micrometers indiameter. In some embodiments, nanoparticles are composed primarily ofmetal, including but not limited to gold, lead, and zinc. In someembodiments, nanoparticles are composed of inorganic salts, includingbut limited to cadmium selenide, zinc telluride, and silicon nitride. Insome embodiments, nanoparticles are uniform in composition. In someembodiments, nanoparticles comprise both core and shell regions ofdiffering composition.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

A typical test to determine whether a compound is a STING agonist is topresent the compound to a wild-type human or animal cell line and in thecorresponding cell line in which the STING coding gene has beengenetically inactivated (e.g. a homozygous STING knockout cell line). ASTING agonist will induce Type I interferons in the wild-type cells butwill not induce Type I interferons in the cells in which the STINGcoding gene has been inactivated.

As used herein, the term “STING agonist” is used to refer to a compoundthat induces activation of STING-dependent pathways at least aseffectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 2-foldas effectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 5-foldas effectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 10-foldas effectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 20-foldas effectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 50-foldas effectively as bis-3′,5′ c-di-GMP. In some embodiments, the STINGagonist induces activation of STING-dependent pathways at least 100-foldas effectively as bis-3′,5′ c-di-GMP.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, “treating,” “treatment,” and the like means amelioratinga disease, so as to reduce, ameliorate, or eliminate its cause, itsprogression, its severity, or one or more of its symptoms, or otherwisebeneficially alter the disease in a subject. Reference to “treating,” or“treatment” of a patient is intended to include prophylaxis. Treatmentmay also be preemptive in nature, i.e., it may include prevention ofdisease in a subject exposed to or at risk for the disease. Preventionof a disease may involve complete protection from disease, for exampleas in the case of prevention of infection with a pathogen, or mayinvolve prevention of disease progression, for example from prediabetesto diabetes. For example, prevention of a disease may not mean completeforeclosure of any effect related to the diseases at any level, butinstead may mean prevention of the symptoms of a disease to a clinicallysignificant or detectable level. Prevention of diseases may also meanprevention of progression of a disease to a later stage of the disease.

The term “patient” is generally synonymous with the term “subject” andincludes all mammals including humans. Examples of patients includehumans, livestock such as cows, goats, sheep, pigs, and rabbits, andcompanion animals such as dogs, cats, rabbits, and horses. Preferably,the patient is a human.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can be administered with the use of adrug delivery vehicle.

As used herein, alone or in combination, the term “drug deliveryvehicle” refers to a molecular assembly that comprises apharmaceutically active compound and a second component that conferspharmacokinetic benefits.

In some embodiments, the drug delivery vehicle comprises apharmaceutically active compound and a targeting moiety.

As used herein, alone or in combination, the term “targeting moiety” isused to describe a moiety that assists in the delivery of thepharmaceutically active compound to a biochemical target. In someembodiments, the pharmaceutically active compound is covalently linkedto the targeting moiety. In some embodiments, the pharmaceuticallyactive compound is covalently linked to the targeting moiety with achemically labile bond. Non-limiting examples of chemically labile bondsused for this purpose include acetals, ketals, esters, and amides. Insome embodiments, the pharmaceutically active compound is noncovalentlyassociated with the targeting moiety.

In some embodiments, the targeting moiety is a biomolecule. Biomoleculesthat can be used as a targeting moiety include, but is not limited to,peptides, proteins, antibodies, nucleic acids, and naturally occurringhormones, cofactors, signalling molecules, and enzyme substrates. Insome embodiments, the targeting moiety is a synthetic analogue of abiomolecule. In some embodiments, the targeting moiety is a syntheticmolecule. In some embodiments, the targeting moiety is a nanoparticle.

In some embodiments, the drug delivery vehicle comprises apharmaceutically active compound and a container moiety.

As used herein, alone or in combination, the term “container moiety”refers to a molecule or supramolecular structure that partially orcompletely encloses a pharmaceutically active compound.

In some embodiments, the container moiety is a single molecule. In someembodiments, the container moiety is a nanoparticle.

In some embodiments, the container moiety is a supramolecular assemblyof noncovalently associated molecules. In some embodiments, thecontainer moiety is a liposome. In some embodiments, the containermoiety is a micelle. In some embodiments, the container moiety is avesicle. In some embodiments, the container moiety comprisesphospholipids.

In some embodiments, the pharmaceutically active compound is fullycontained within the container moiety. In some embodiments, thepharmaceutically active compound is partially contained within thecontainer moiety. In some embodiments, the pharmaceutically activecompound is noncovalently associated with the container moiety.

In some embodiments, the drug delivery vehicle comprises a selectivitymoiety.

As used herein, alone or in combination, the term “selectivity moiety”refers to a molecule that confers selective delivery to a certain regionof the body of a subject. In some embodiments, the selectivity moietyselectively delivers the drug delivery vehicle to an organ of interest.In some embodiments, the selectivity moiety selectively delivers thedrug delivery vehicle to a tumor. In some embodiments, the selectivitymoiety improves transport across biological membranes. In someembodiments, the selectivity moiety improves transport from thegastrointestinal tract into the bloodstream. In some embodiments, theselectivity moiety improves transport across the blood-brain barrier. Aselectivity moiety may also be a targeting moiety, and vice versa.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present invention includes compounds listed above in the formof salts, including acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent invention contemplates sodium, potassium, magnesium, and calciumsalts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

While it may be possible for the compounds of the subject invention tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. The pharmaceutical compositionsdisclosed herein may be manufactured in any manner known in the art,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orcompression processes.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject invention or a pharmaceutically acceptable salt, ester,tautomer, amide, prodrug or solvate thereof (“active ingredient”) withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both and then, if necessary, shapingthe product into the desired formulation.

Formulations of the compounds disclosed herein suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In certain embodiments, parenteral administration is facilitated withliposomes. Liposomes are vesicles formed from one or more layers ofphospholipid. Liposomes comprise both hydrophilic and hydrophobicregions, and can be used to improve the pharmacokinetic properties of aformulation. In some embodiments, the liposomes can comprise one or moreadditional components, such as peptidoglycan, lipopeptide,lipopolysaccharide, phosphorylated lipid A, acylatedphosphatidylcholine, acylated glycerol, oligonucleotides, ceramides,retinoic acid, quaternary ammonium salts, anionic, cationic, andnonionic surfactants, lipoteichoic acid, resiquimod, imiquimod, andflagellin.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds disclosed herein may be administered topically, thatis by non-systemic administration. This includes the application of acompound disclosed herein externally to the epidermis or the buccalcavity and the instillation of such a compound into the ear, eye andnose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient for topical administration maycomprise, for example, from 0.001% to 10% w/w (by weight) of theformulation. In certain embodiments, the active ingredient may compriseas much as 10% w/w. In other embodiments, it may comprise less than 5%w/w. In certain embodiments, the active ingredient may comprise from 2%w/w to 5% w/w. In other embodiments, it may comprise from 0.1% to 1% w/wof the formulation.

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form, in for example, capsules, cartridges,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations described above may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The compounds can be administered in various modes, e.g. orally,topically, or by injection. The precise amount of compound administeredto a patient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. Also, the route of administrationmay vary depending on the condition and its severity.

In certain instances, it may be appropriate to administer at least oneof the compounds described herein (or a pharmaceutically acceptablesalt, ester, tautomer, or prodrug thereof) in combination with anothertherapeutic agent. By way of example only, if one of the side effectsexperienced by a patient upon receiving one of the compounds herein ishypertension, then it may be appropriate to administer ananti-hypertensive agent in combination with the initial therapeuticagent. Or, by way of example only, the therapeutic effectiveness of oneof the compounds described herein may be enhanced by administration ofan adjuvant (i.e., by itself the adjuvant may only have minimaltherapeutic benefit, but in combination with another therapeutic agent,the overall therapeutic benefit to the patient is enhanced). Or, by wayof example only, the benefit of experienced by a patient may beincreased by administering one of the compounds described herein withanother therapeutic agent (which also includes a therapeutic regimen)that also has therapeutic benefit. By way of example only, in atreatment for diabetes involving administration of one of the compoundsdescribed herein, increased therapeutic benefit may result by alsoproviding the patient with another therapeutic agent for diabetes. Inany case, regardless of the disease, disorder or condition beingtreated, the overall benefit experienced by the patient may simply beadditive of the two therapeutic agents or the patient may experience asynergistic benefit.

In any case, the multiple therapeutic agents (at least one of which is acompound disclosed herein) may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may be any duration of time ranging from a few minutes tofour weeks.

Thus, in another aspect, certain embodiments provide methods fortreating STING-mediated disorders in a human or animal subject in needof such treatment comprising administering to said subject an amount ofa compound disclosed herein effective to reduce or prevent said disorderin the subject, in combination with at least one additional agent forthe treatment of said disorder that is known in the art. In a relatedaspect, certain embodiments provide therapeutic compositions comprisingat least one compound disclosed herein in combination with one or moreadditional agents for the treatment of STING-mediated disorders.

Besides being useful for human treatment, certain compounds andformulations disclosed herein may also be useful for veterinarytreatment of companion animals, exotic animals and farm animals,including mammals, rodents, and the like. More preferred animals includehorses, dogs, and cats.

Autoimmune Disorders

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment of autoimmunediseases and disorders. In some embodiments, the autoimmune disorder isselected from systemic lupus erythmatosis, psoriasis, insulin-dependentdiabetes, dermatomyositis, and Sjogren's syndrome. In some embodiments,the autoimmune disorder is multiple sclerosis.

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment of immune systemdeficiencies or defects.

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment of microbial orviral infections. In some embodiments, the viral infection is hepatitis.

Inflammation and Inflammatory Disease

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment of inflammation oran inflammatory disease or disorder. In some embodiments, theinflammatory disease or disorder is selected from musculoskeletalinflammation, vascular inflammation, neural inflammation, digestivesystem inflammation, inflammation of the reproductive system, and ocularinflammation.

In some embodiments, the inflammatory disease or disorder is selectedfrom arthritis, tendonitis, synovitis, tenosynovitis, bursitis,fibrositis (fibromyalgia), epicondylitis, myositis, osteitis,blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis,keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis,uveitis, encephalitis, Guillain-Barre syndrome, meningitis,neuromyotonia, narcolepsy, multiple sclerosis, myelitis, schizophrenia,arthrosclerosis, arthritis, phlebitis, vasculitis, lymphangitis,cholangitis, cholecystitis, enteritis, enterocolitis, gastritis,gastroenteritis, inflammatory bowel disease (such as Crohn's disease andulcerative colitis), ileitis, proctitis, cervicitis, chorioamnionitis,endometritis, epididymitis, omphalitis, oophoritis, orchitis,salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, andvulvodynia.

In some embodiments, the inflammatory disease or disorder is selectedfrom appendicitis, dermatitis, dermatomyositis, endocarditis,fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa,iritis, laryngitis, mastitis, myocarditis, nephritis, otitis,pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis,pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi,transplant rejection (involving organs such as kidney, liver, heart,lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel,skin allografts, skin homografts, and heart valve xengrafts, sewrumsickness, and graft vs host disease), acute pancreatitis, chronicpancreatitis, acute respiratory distress syndrome, Sexary's syndrome,congenital adrenal hyperplasis, nonsuppurative thyroiditis,hypercalcemia associated with cancer, pemphigus, bullous dermatitisherpetiformis, severe erythema multiforme, exfoliative dermatitis,seborrheic dermatitis, seasonal or perennial allergic rhinitis,bronchial asthma, contact dermatitis, astopic dermatitis, drughypersensistivity reactions, allergic conjunctivitis, keratitis, herpeszoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, opticneuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonarytuberculosis chemotherapy, idiopathic thrombocytopenic purpura inadults, secondary thrombocytopenia in adults, acquired (autroimmine)hemolytic anemia, leukemia and lymphomas in adults, acute leukemia ofchildhood, regional enteritis, autoimmune vasculitis, multiplesclerosis, chronic obstructive pulmonary disease, solid organ transplantrejection, sepsis. Preferred treatments include treatment of transplantrejection, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemiclupus erythematosis, psoriasis, chronic pulmonary disease, andinflammation accompanying infectious conditions (e.g., sepsis).

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful for the treatment of autoimmunedisease or disorder having an inflammatory component. In someembodiments, the autoimmune disease or disorder is selected from acutedisseminated alopecia universalise, Behcet's disease, Chagas' disease,chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosingspondylitis, aplastic anemia, hidradenitis suppurativa, autoimmunehepatitis, autoimmune oophoritis, celiac disease, Crohn's disease,diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome,Grave's disease, Guillain-Barre syndrome, Hashimoto's disease,Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus,microscopic colitis, microscopic polyarteritis, mixed connective tissuedisease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonussyndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritisnodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehemolytic anemia, interstitial cystitis, lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful for the treatment of aneurodegenerative disease or disorder. In some embodiments, theneurodegenerative disease or disorder is chosen from Parkinson'sdisease, Alzheimer's disease, Huntington's disease, and amyotrophiclateral sclerosis.

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be administered before, during, or afteradministration of a vaccine. The benefits afforded by thecoadministration may include: enhanced efficacy of the vaccine, reducedtoxicity of the vaccine, or reduced side effects of the vaccine. In someembodiments, the vaccine can comprise inactivated or attenuated bacteriaor viruses. In some embodiments, the vaccine can comprise purifiedantigens. In some embodiments, the vaccine can comprise live viral orbacterial delivery vectors that have been recombinantly engineered toexpress or secrete an antigen. In some embodiments, the vaccine cancomprise antigen presenting cells that are loaded with antigen. In someembodiments, the vaccine may comprise an inactivated tumor cell.

Cancer

In some embodiments, the compounds and pharmaceutical compositions ofthe present disclosure may be useful in the treatment or prevention ofcancer.

In some embodiments, the compounds of the present disclosure may be usedto prevent or treat cancer, wherein the cancer is one or a variant ofAcute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma andLymphoma), Anal Cancer, Appendix Cancer, Atypical Teratoid/RhabdoidTumor, Basal Cell Carcinoma, Bile Duct Cancer (including Extrahepatic),Bladder Cancer, Bone Cancer (including Osteosarcoma and MalignantFibrous Histiocytoma), Brain Tumor (such as Astrocytomas, Brain andSpinal Cord Tumors, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors,Craniopharyngioma, Ependymoblastoma, Ependymoma, Medulloblastoma,Medulloepithelioma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, BasalCell Carcinoma, Bile Duct Cancer (including Extrahepatic), BladderCancer, Bone Cancer (including Osteosarcoma and Malignant FibrousHistiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary, CentralNervous System (such as Atypical Teratoid/Rhabdoid Tumor, EmbryonalTumors and Lymphoma), Cervical Cancer, Childhood Cancers, Chordoma,Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma (Mycosis Fungoides andSezary Syndrome), Duct, Bile (Extrahepatic), Ductal Carcinoma In Situ(DCIS), Embryonal Tumors (Central Nervous System), Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer(like Intraocular Melanoma, Retinoblastoma), Fibrous Histiocytoma ofBone (including Malignant and Osteosarcoma) Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalStromal Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia,Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors (Endocrine, Pancreas), KaposiSarcoma, Kidney (including Renal Cell), Langerhans Cell Histiocytosis,Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), AcuteMyeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML),Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSezary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Lip and, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer (such as Epithelial, GermCell Tumor, and Low Malignant Potential Tumor), Pancreatic Cancer(including Islet Cell Tumors), Papillomatosis, Paraganglioma, ParanasalSinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, PrimaryCentral Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer,Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional CellCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma(like Ewing Sarcoma Family of Tumors, Kaposi, Soft Tissue, Uterine),Sezary Syndrome, Skin Cancer (such as Melanoma, Merkel Cell Carcinoma,Nonmelanoma), Small Cell Lung Cancer, Small Intestine Cancer, SoftTissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer withOccult Primary, Metastatic, Stomach (Gastric) Cancer, SupratentorialPrimitive Neuroectodermal Tumors, T-Cell Lymphoma (Cutaneous, MycosisFungoides and Sezary Syndrome), Testicular Cancer, Throat Cancer,Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancerof the Renal Pelvis and Ureter, Trophoblastic Tumor (Gestational),Unknown Primary, Unusual Cancers of Childhood, Ureter and Renal Pelvis,Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial,Uterine Sarcoma, Waldenström Macroglobulinemia or Wilms Tumor.

In some embodiments, methods described herein are used to treat adisease condition comprising administering to a subject in need thereofa therapeutically effective amount of a compound of Formula I orpharmaceutically acceptable salt thereof, wherein the condition iscancer which has developed resistance to chemotherapeutic drugs and/orionizing radiation.

Adjuvants for Vaccines

In some embodiments of the present invention, there is provided acompound of formula (Ia), or a pharmaceutically acceptable salt thereoffor use as vaccine adjuvants.

In some embodiments there is further provided a vaccine adjuvantcomprising a compound of formula (Ia), or a pharmaceutically acceptablesalt thereof.

In some embodiments there is further provided an immunogenic compositioncomprising an antigen or antigen composition and a compound of formula(Ia), or a pharmaceutically acceptable salt thereof.

In some embodiments there is further provided an immunogenic compositioncomprising an antigen or antigen composition and a compound of formula(Ia), or a pharmaceutically acceptable salt thereof for use in thetreatment or prevention of disease.

In some embodiments there is further provided the use of a compound offormula (Ia), or a pharmaceutically acceptable salt thereof, for themanufacture of an immunogenic composition comprising an antigen orantigen composition, for the treatment or prevention of disease. In someembodiments there is further provided a method of treating or preventingdisease comprising the administration to a human subject suffering fromor susceptible to disease, an immunogenic composition comprising anantigen or antigen composition and a compound of formula (Ia), or apharmaceutically acceptable salt thereof.

In some embodiments there is further provided a vaccine compositioncomprising an antigen or antigen composition and a compound of formula(Ia), or a pharmaceutically acceptable salt thereof for use in thetreatment or prevention of disease. In some embodiments there is furtherprovided a vaccine composition comprising an antigen or antigencomposition and a compound of formula (Ia), or a pharmaceuticallyacceptable salt thereof for use in the treatment or prevention ofdisease

In some embodiments there is further provided the use of a compound offormula (I), or a pharmaceutically acceptable salt thereof, for themanufacture of a vaccine composition comprising an antigen or antigencomposition, for the treatment or prevention of disease.

In some embodiments there is further provided a method of treating orpreventing disease comprising the administration to a human subjectsuffering from or susceptible to disease, a vaccine compositioncomprising an antigen or antigen composition and a compound of formula(I), or a pharmaceutically acceptable salt thereof.

The compounds of formula (Ia) and pharmaceutically acceptable saltsthereof may also be formulated with vaccines as adjuvants to modulatetheir activity. Such compositions may contain one or more antibodies, orone or more antibody fragments or an antigenic component including butnot limited to protein, DNA, live or dead bacteria and/or viruses orvirus-like particles. Such compositions may also contain one or morecomponents with adjuvant activity including but not limited to aluminiumsalts, oil and water emulsions, heat shock proteins, lipid Apreparations and derivatives, glycolipids, other TLR agonists such asCpG DNA or similar agents, cytokines such as GM-CSF or IL-12 or similaragents.

Combinations and Combination Therapy

The compounds of the present invention can be used, alone or incombination with other pharmaceutically active compounds, to treatconditions such as those previously described hereinabove. Thecompound(s) of the present invention and other pharmaceutically activecompound(s) can be administered simultaneously (either in the samedosage form or in separate dosage forms) or sequentially. Accordingly,in one embodiment, the present invention comprises methods for treatinga condition by administering to the subject a therapeutically-effectiveamount of one or more compounds of the present invention and one or moreadditional pharmaceutically active compounds.

In another embodiment, there is provided a pharmaceutical compositioncomprising one or more compounds of the present invention, one or moreadditional pharmaceutically active compounds, and a pharmaceuticallyacceptable carrier.

In another embodiment, the one or more additional pharmaceuticallyactive compounds is selected from anti-cancer drugs, anti-proliferativedrugs, and anti-inflammatory drugs.

STING agonist compositions described herein are also optionally used incombination with other therapeutic reagents that are selected for theirtherapeutic value for the condition to be treated. In general, thecompounds described herein and, in embodiments where combination therapyis employed, other agents do not have to be administered in the samepharmaceutical composition and, because of different physical andchemical characteristics, are optionally administered by differentroutes. The initial administration is generally made according toestablished protocols and then, based upon the observed effects, thedosage, modes of administration and times of administration subsequentlymodified. In certain instances, it is appropriate to administer a STINGagonist compound, as described herein, in combination with anothertherapeutic agent. By way of example only, the therapeutic effectivenessof a STING agonist is enhanced by administration of another therapeuticagent (which also includes a therapeutic regimen) that also hastherapeutic benefit. Regardless of the disease, disorder or conditionbeing treated, the overall benefit experienced by the patient is eithersimply additive of the two therapeutic agents or the patient experiencesan enhanced (i.e., synergistic) benefit. Alternatively, if a compounddisclosed herein has a side effect, it may be appropriate to administeran agent to reduce the side effect; or the therapeutic effectiveness ofa compound described herein may be enhanced by administration of anadjuvant.

Therapeutically effective dosages vary when the drugs are used intreatment combinations. Methods for experimentally determiningtherapeutically effective dosages of drugs and other agents for use incombination treatment regimens are documented methodologies. Combinationtreatment further includes periodic treatments that start and stop atvarious times to assist with the clinical management of the patient. Inany case, the multiple therapeutic agents (one of which is a STINGagonist as described herein) may be administered in any order, orsimultaneously. If simultaneously, the multiple therapeutic agents areoptionally provided in a single, unified form, or in multiple forms (byway of example only, either as a single pill or as two separate pills).

In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses optionally varies from more than zeroweeks to less than twelve weeks.

In addition, the combination methods, compositions and formulations arenot to be limited to the use of only two agents, the use of multipletherapeutic combinations are also envisioned. It is understood that thedosage regimen to treat, prevent, or ameliorate the condition(s) forwhich relief is sought, is optionally modified in accordance with avariety of factors. These factors include the disorder from which thesubject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, the dosage regimen actually employedvaries widely, in some embodiments, and therefore deviates from thedosage regimens set forth herein.

The pharmaceutical agents which make up the combination therapydisclosed herein are optionally a combined dosage form or in separatedosage forms intended for substantially simultaneous administration. Thepharmaceutical agents that make up the combination therapy areoptionally also administered sequentially, with either agent beingadministered by a regimen calling for two-step administration. Thetwo-step administration regimen optionally calls for sequentialadministration of the active agents or spaced-apart administration ofthe separate active agents. The time between the multiple administrationsteps ranges from a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent.

In another embodiment, a STING agonist is optionally used in combinationwith procedures that provide additional benefit to the patient. A STINGagonist and any additional therapies are optionally administered before,during or after the occurrence of a disease or condition, and the timingof administering the composition containing a STING agonist varies insome embodiments. Thus, for example, a STING agonist is used as aprophylactic and is administered continuously to subjects with apropensity to develop conditions or diseases in order to prevent theoccurrence of the disease or condition. A STING agonist and compositionsare optionally administered to a subject during or as soon as possibleafter the onset of the symptoms. While embodiments of the presentinvention have been shown and described herein, it will be obvious tothose skilled in the art that such embodiments are provided by way ofexample only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.It should be understood that in some embodiments of the inventionvarious alternatives to the embodiments described herein are employed inpracticing the invention.

A STING agonist can be used in combination with anti-cancer drugs,including but not limited to the following classes: alkylating agents,anti-metabolites, plant alkaloids and terpenoids, topoisomeraseinhibitors, cytotoxic antibiotics, angiogenesis inhibitors and tyrosinekinase inhibitors.

For use in cancer and neoplastic diseases a STING agonist may beoptimally used together with one or more of the following non-limitingexamples of anti-cancer agents:

-   1) alkylating agents, including but not limited to carmustine,    chlorambucil (LEUKERAN), cisplatin (PLATIN), carboplatin    (PARAPLATIN), oxaliplatin (ELOXATIN), streptozocin (ZANOSAR),    busulfan (MYLERAN), dacarbazine, ifosfamide, lomustine (CCNU),    melphalan (ALKERAN), procarbazine (MATULAN), temozolomide (TEMODAR),    thiotepa, and cyclophosphamide (ENDOXAN);-   2) anti-metabolites, including but not limited to cladribine    (LEUSTATIN), mercaptopurine (PURINETHOL), thioguanine, pentostatin    (NIPENT), cytosine arabinoside (cytarabine, ARA-C), gemcitabine    (GEMZAR), fluorouracil (5-FU, CARAC), capecitabine (XELODA),    leucovorin (FUSILEV), methotrexate (RHEUMATREX), raltitrexed;-   3) antimitotics, which are often plant alkaloids and terpenoids, or    derivateves thereof, including but not limited to taxanes such as    docetaxel (TAXITERE) and paclitaxel (ABRAXANE, TAXOL); vinca    alkaloids such as vincristine (ONCOVIN), vinblastine, vindesine, and    vinorelbine (NAVELBINE);-   4) topoisomerase inhibitors, including but not limited to    camptothecin (CTP), irinotecan (CAMPTOSAR), topotecan (HYCAMTIN),    teniposide (VUMON), and etoposide (EPOSIN);-   5) cytotoxic antibiotics, including but not limited to actinomycin D    (dactinomycin, COSMEGEN), bleomycin (BLENOXANE) doxorubicin    (ADRIAMYCIN), daunorubicin (CERUBIDINE), epirubicin (ELLENCE),    fludarabine (FLUDARA), idarubicin, mitomycin (MITOSOL), mitoxantrone    (NOVANTRONE), plicamycin;-   6) aromatase inhibitors, including but not limited to    aminoglutethimide, anastrozole (ARIMIDEX), letrozole (FEMARA),    vorozole (RIVIZOR), exemestane (AROMASIN);-   7) angiogenesis inhibitors, including but not limited to genistein,    sunitinib (SUTENT) and bevacizumab (AVASTIN);-   8) anti-steroids and anti-androgens such as aminoglutethimide    (CYTADREN), bicalutamide (CASODEX), cyproterone, flutamide    (EULEXIN), nilutamide (NILANDRON);-   9) tyrosine kinase inhibitors, including but not limited to imatinib    (GLEEVEC), erlotinib (TARCEVA), lapatininb (TYKERB), sorafenib    (NEXAVAR), and axitinib (INLYTA);-   10) mTOR inhibitors such as everolimus, temsirolimus (TORISEL), and    sirolimus;-   11) monoclonal antibodies such as trastuzumab (HERCEPTIN) and    rituximab (RITUXAN);-   12) other agents, such as amsacrine; Bacillus Calmette-Guerin    (B-C-G) vaccine; buserelin (ETILAMIDE); chloroquine (ARALEN);    clodronate, pamidronate, and other bisphosphonates; colchicine;    demethoxyviridin; dichloroacetate; estramustine; filgrastim    (NEUPOGEN); fludrocortisone (FLORINEF); goserelin (ZOLADEX);    interferon; leucovorin; leuprolide (LUPRON); levamisole; lonidamine;    mesna; metformin; mitotane (o,p′-DDD, LYSODREN); nocodazole;    octreotide (SANDOSTATIN); perifosine; porfimer (particularly in    combination with photo- and radiotherapy); suramin; tamoxifen;    titanocene dichloride; tretinoin; anabolic steroids such as    fluoxymesterone (HALOTESTIN); estrogens such as estradiol,    diethylstilbestrol (DES), and dienestrol; progestins such as    medroxyprogesterone acetate (MPA) and megestrol; and testosterone.

For use in treatment of multiple sclerosis, a STING agonist may beoptimally used together with one or more of the following non-limitingexamples of agents: glatiramer, corticosteroids, muscle relaxants suchas tizanidine (ZANAFLEX) or baclofen (LIORESAL), agents to reducefatigue, such as amantadine (SYMMETREL), modafinil (PROVIGIL), andagents to alleviate depression, pain, and bladder or bowel controlproblems.

In some embodiments, a STING agonist may be optimally used together withone or more of the following non-limiting examples of immune checkpointinhibitors: CTLA-4, PD-1, Tim-3, Vista, BTLA, LAG-3 and TIGIT pathwayantagonists; PD-1 pathway blocking agents; PD-L1 inhibitors; includingwithout limitation anti-PD-1 antibodies nivolumab, pembrolizumab orpidilizumab; PD-1 inhibitor AMP-224; anti-CTLA-4 antibody ipilimumab;and anti-PD-L1 antibodies BMS-936559, MPDL3280A, MEDI4736, and avelumab.

In some embodiments, a STING agonist may be optimally used together withone or moe of the following non-limiting examples of antibodytherapeutical agents: muromonab-CD3, infliximab (REMICADE), adalimumab(HUMIRA), omalizumab (XOLAIR), daclizumab (ZENAPAX), rituximab(RITUXAN), ibritumomab (ZEVALIN), tositumomab (BEXXAR), cetuximab(ERBITUX), trastuzumab (HERCEPTIN), ADCETRIS, alemtuzumab (CAMPATH-1H),Lym-1 (ONCOLYM), ipilimumab (YERVOY), vitaxin, bevacizumab (AVASTIN),and abciximab (REOPRO).

Where a subject is suffering from or at risk of suffering from aninflammatory condition, a STING agonist compound described herein isoptionally used together with one or more agents or methods for treatingan inflammatory condition in any combination. Therapeuticagents/treatments for treating an autoimmune and/or inflammatorycondition include, but are not limited to any of the following examples:

-   1) corticosteroids, including but not limited to cortisone,    dexamethasone, and methylprednisolone;-   2) nonsteroidal anti-inflammatory drugs (NSAIDs), including but not    limited to ibuprofen, naproxen, acetaminophen, aspirin, fenoprofen    (NALFON), flurbiprofen (ANSAID), ketoprofen, oxaprozin (DAYPRO),    diclofenac sodium (VOLTAREN), diclofenac potassium (CATAFLAM),    etodolac (LODINE), indomethacin (INDOCIN), ketorolac (TORADOL),    sulindac (CLINORIL), tolmetin (TOLECTIN), meclofenamate (MECLOMEN),    mefenamic acid (PONSTEL), nabumetone (RELAFEN) and piroxicam    (FELDENE);-   3) immunosuppressants, including but not limited to methotrexate    (RHEUMATREX), leflunomide (ARAVA), azathioprine (IMURAN),    cyclosporine (NEORAL, SANDIMMUNE), tacrolimus and cyclophosphamide    (CYTOXAN);-   4) CD20 blockers, including but not limited to rituximab (RITUXAN);-   5) Tumor Necrosis Factor (TNF) blockers, including but not limited    to etanercept (ENBREL), infliximab (REMICADE) and adalimumab    (HUMIRA);-   6) interleukin-1 receptor antagonists, including but not limited to    anakinra (KINERET);-   7) interleukin-6 inhibitors, including but not limited to    tocilizumab (ACTEMRA);-   8) interleukin-17 inhibitors, including but not limited to AIN457;-   9) Janus kinase inhibitors, including but not limited to    tasocitinib; and-   10) syk inhibitors, including but not limited to fostamatinib.

The compositions may also be administered in combination withradiotherapy, surgical therapy, immunotherapy, cryotherapy, genetherapy, or any other therapy known for use by the person of ordinaryskill in the art.

Combination therapies may be applied during the same time period, orsequentially, or with overlapping time intervals. Nonsequentialcombination therapy may alternate between the two or more therapies. Insome situations, it may be desirable to extend the time period foradministration of a certain therapy. In some situations, it may bedesirable to shorten or prolong the time period between therapies.

List of Abbreviations

DCI=4,5-dicyanoimidazole;DDTT=3-((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione;DMAP=4-Dimethylaminopyridine;DMOCP=2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane;DMTr=dimethoxytrityl=(4-methoxyphenyl)₂(phenyl)methyl;Piv=pivaloyl=(CH₃)₃C—C(═O)—; NaOH=sodium hydroxide; M=molar;mL=milliliter; h=hour; min.=minute; HCl=hydrogen chloride; H₂O=water;MS=mass spectrometry; ES+=electrospray positive ionization;¹H-NMR=proton nuclear magnetic resonance; ³¹P-NMR=phosphorous nuclearmagnetic resonance; MHz=megahertz; H=hydrogen; RT=room temperature; °C.=Celsius; Br₂=bromine; NaHSO₃=sodium bisulfite;NMP=N-Methyl-2-pyrrolidone; MW=microwave; KF=potassium fluoride;Pd(dppf)Cl₂=[1,1-bis(diphenylphosphino)ferrocene]palladium(II)dichloride; PE=petroleum ether; EtOAc=ethyl acetate; EA=ethyl acetate;CDCl₃=deuterated chloroform; DMSO-d₆=dimethyl sulfoxide deuterated-6;CD₃CN=deuterated acetonitrile; MeOH=methanol; D₂O=deuterated water;prep-HPLC=preparative high pressure liquid chromatography, also known aspreparative high performance liquid chromatography; DMSO=dimethylsulfoxide; MeCN=acetonitrile; NH₃=ammonia; NH₄OH=ammonium hydroxide;NIS=N-iodosuccinimide; DMF=dimethylformamide; K₃PO₄=potassium phosphate,tribasic; N₂=nitrogen; Py=pyridine; THF=tetrahydrofuran;TEA=triethylamine; TBSCl=tert-butyldimethylsilyl chloride;TEAB=tetraethyl ammonium bicarbonate; TMSCl=trimethylsilyl chloride;

TFA=trifluoroacetic acid; DCM=dichloromethane; K₂CO₃=potassiumcarbonate; ul=microliter.

General Synthetic Methods for Preparing Compounds

The following schemes can be used to practice the present invention.

Compounds disclosed herein can be synthesized using the followinggeneral synthetic procedure set forth in Scheme I, employing variationswhich will be apparent to those skilled in the art. Nucleotide ornucleotide analogue 101, having an unprotected 5′ hydroxyl, andnucleotide or nucleotide analogue 102, having a phosphoramidite moietyat its 2′ hydroxyl, are coupled with pyridinium fluoride. Theintermediate phosphite is oxidatively sulfurized with DDTT, giving amixture of R_(P) and S_(P) diastereoisomers at the phosphorothioatecenter.

In a third step, dichloroacetic acid is used to remove the DMTr group onthe fragment derived from 102. Intermediate 103 is then cyclized withDMOCP, then oxidatively sulfurized with Beaucage reagent(3H-1,2-benzodithiol-3-one 1,1-dioxide). Stereochemistry at the newlyformed phosphorothioate center can result in a mixture of R_(p) andS_(p) stereoisomers. Based on literature precedent (Gaffney, B. L. etal. Org. Lett. 2010, 12(14), 3269-3271; Zhao, J. et al. NucleosidesNucleotides Nucl Acids 2009, 28, 352-378; Battistini, C. et al.Tetrahedron 1993, 49, 1115-1132.), the stereochemistry at this center isoften, but not always, assigned as R_(p), and is indicated as (R) inScheme 1. Synthesis is completed by removal of the cyanoethyl group of104 with ammonia, followed by removal of the silyl protecting groups of105 with fluoride. Cyclic dinucleotides 106 are obtained as a mixture ofdiastereoisomers, typically assigned as R_(P)R_(P) and S_(P)R_(P) at thephosphorothioate centers based on the above literature precedent. It isalso possible that more than two diastereoisomers may be obtained insome cases. The diastereoisomers can be separated and obtained insubstantially pure form via reverse phase HPLC purification. If desired,the anionic form can be obtained by treatment with ion exchange resin.Without limitation, the disodium salt can be obtained in this fashion.

The invention is further illustrated by the following examples.

INTERMEDIATE A(2R,3R,4R,5R)-2-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite

Step 1

(2R,3R,4S,5R)-2-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol

A mixture of(2R,3R,4S,5R)-2-(4-chloro-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol(7.0 g, 24.5 mmol) and 7 N NH₃ in MeOH (70 mL) was stirred at 110° C.for 16 h in a pressure safe steel vessel. The mixture was cooled to RTand the volatiles were removed under reduced pressure. Ten batches ofthis reaction were run in parallel. The residues were combined andtriturated with MeOH (500 ml) to give the title compound as an off-whitesolid.

Step 2

N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution of the product from the previous step (65.2 g, 245 mmol)in pyridine (1.14 L) at 0° C. was added TMSCl (119.8 g, 1.10 mol, 4.5eq) dropwise over 30 minutes. The mixture was stirred for further 30minutes at 0° C., and BzCl (6 g, 34.9 mmol, 1.5 eq) was then addeddropwise. The resulting mixture was allowed to stir at RT for 16 h,cooled to 0° C. and then quenched with H₂O (200 mL), followed by 25% aq.NH₄OH (500 mL). The volatiles were removed under reduced pressure; theresidue was diluted in H₂O (1.5 L) and extracted with EtOAc (3×2.0 L).The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by SiO₂gel chromatography (DCM:MeOH=20:1) to afford the title compound (60.7 g,0.164 mol, 67% over two steps).

MS(ES⁺) C₁₈H₁₉N₄O₅ requires: 371, found: 370.8 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d₆) δ ppm 11.15 (s, 1H), 8.61 (s, 1H), 8.07 (d, J=7.4 Hz, 2H), 7.74(d, J=3.8 Hz, 1H), 7.61-7.67 (m, 1H), 7.52-7.58 (m, 2H), 6.69 (d, J=3.6Hz, 1H), 6.24 (d, J=6.4 Hz, 1H), 5.38 (d, J=6.4 Hz, 1H), 5.18 (d, J=4.8Hz, 1H), 5.08 (t, J=5.5 Hz, 1H), 4.43 (q, J=6.1 Hz, 1H), 4.09-4.15 (m,1H), 3.93 (q, J=3.6 Hz, 1H), 3.52-3.68 (m, 2H).

Step 3

N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

To a solution of the product from the previous step (60.0 g, 162.0 mmol)in pyridine (420 mL) was added DMTrCl (65.87 g, 194.4 mmol, 1.2 eq). Themixture was stirred at RT for 16 h, diluted with CH₂Cl₂ (1.0 L), washedwith sat NaHCO₃ (2×500 mL), H₂O (500 mL) and brine (500 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified by SiO₂ gel chromatography(5/1 petroleum ether/EtOAc to 100% EtOAc) to afford the title compound(89.3 g, 132.8 mmol, 82%) as a white foam.

Step 4

N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-hydroxy-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide(A-4a) andN-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-3-hydroxy-4-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide(A-4b)

To a mixture of the product from the previous step (55 g, 81.76 mmol)and AgNO₃ (22.92 g, 134.9 mmol, 22.7 mL, 1.65 eq) in THF (400 mL) wasadded TBSCl (78.75 g, 522.48 mmol, 1.76 eq). The reaction mixture wasstirred at RT for 5 h, filtered and concentrated under reduced pressure.The residue was purified by SiO₂ gel chromatography (PetroleumEther/EtOAc=10/1 to 2/1) to afford A-4a (70 g, 88.9 mmol, 54.4%) andA-4b (7 g, 8.89 mmol, 5.4%).

A-4a: ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.16 (br s, 1H), 8.58 (s, 1H),8.07 (d, J=7.7 Hz, 2H), 7.52-7.67 (m, 4H), 7.42 (d, J=7.7 Hz, 2H),7.22-7.32 (m, 7H), 6.88 (d, J=8.8 Hz, 4H), 6.69 (d, J=3.8 Hz, 1H), 6.30(d, J=5.4 Hz, 1H), 5.12 (d, J=5.8 Hz, 1H), 4.59 (t, J=5.3 Hz, 1H),4.07-4.21 (m, 2H), 3.73 (s, 6H), 3.28 (br s, 2H), 0.75 (s, 9H), −0.04(s, 3H), −0.16 (s, 3H).

A-4b: ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.76 (brs, 1H), 8.59 (s, 1H),8.07 (d, J=7.8 Hz, 2H), 7.61-7.66 (m, 2H), 7.52-7.57 (m, 2H), 7.38 (brd, J=7.8 Hz, 2H), 7.19-7.32 (m, 7H), 6.86 (d, J=8.7 Hz, 4H), 6.68 (d,J=3.6 Hz, 1H), 6.21 (d, J=5.6 Hz, 1H), 5.38 (br d, J=5.9 Hz, 1H), 4.57(br d, J=5.1 Hz, 1H), 4.34 (t, J=4.4 Hz, 1H), 4.00 (br d, J=4.1 Hz, 1H),3.72 (s, 6H), 3.30-3.39 (m, 1H), 3.12-3.16 (m, 1H), 3.14 (br dd, J=4.7,10.4 Hz, 1H), 0.84 (s, 9H), 0.08 (s, 3H), 0.03 (s, 3H).

Step 5

(2R,3R,4R,5R)-2-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite

To a solution of A-4b (10.0 g, 12.71 mmol) in CH₂Cl₂ (100 mL) were addedwere added 3-((bis(diisopropylamino)phosphanyl)-oxy)propanenitrile (4.21g, 14 mmol, 1.1 eq) and DCI (2.25 g, 19.07 mmol, 1.5 eq). The mixturewas stirred at RT for 5 h, diluted with CH₂Cl₂ (100 mL) and washed withsat NaHCO₃ (3×100 mL). The organic layer was dried over Na₂SO₄, filteredand concentrated under reduced pressure. The residue was purified bySiO₂ gel chromatography (petroleum ether/EtOAc=10/1 to 3/1; 0.5% TEA) toafford the title compound (10.0 g, 9.72 mmol, 76%) as a white foam.

¹H-NMR (400 MHz, CD₃CN) δ ppm 9.23 (br s, 1H), 8.52 (br s, 1H), 8.01 (brd, J=5.8 Hz, 2H), 7.60-7.68 (m, 1H), 7.51-7.58 (m, 2H), 7.40-7.50 (m,3H), 7.18-7.34 (m, 7H), 6.79-6.92 (m, 5H), 6.34-6.48 (m, 1H), 4.97-4.70(m, 1H), 4.63-4.48 (m, 1H), 4.13 (br d, J=3.9 Hz, 1H), 3.84-3.69 (m,7H), 3.62-3.41 (m, 4H), 3.27-3.15 (m, 1H), 2.58 (t, J=6.2 Hz, 1H), 2.41(t, J=6.2 Hz, 1H), 1.12-1.03 (m, 9H), 0.91-0.84 (m, 12H), 0.13 (d,J=16.2 Hz, 3H), 0.05 (s, 3H); ³¹P NMR (162 MHz, CD₃CN) δ ppm 149.92,149.53.

INTERMEDIATE B(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

Step 1

(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-ylhydrogen phosphonate

Phosphorous acid (15.63 g, 190.6 mmol, 15 eq) was co-evaporated threetimes with anhydrous pyridine (5 mL) and then dissolved in anhydrouspyridine (75 mL) upon heating to ca. 45° C. The mixture was allowed tocool to RT. A-4a (10.0 g, 12.7 mmol) was added and the mixture wascooled to 0° C. Pivaloyl chloride (15.32 g, 127.07 mmol, 10.0 eq) wasslowly added at 0° C. and the resulting mixture was allowed to warm toRT and stirred for 16 h. The reaction mixture was then quenched by 1 Maq. TEAB (100 mL) and extracted with EtOAc (3×1000 mL). The combinedorganic layers were washed with 0.5 M aq. TEAB (100 m L), and brine(1000 mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified by SiO₂ gel chromatography(CH₂Cl₂/MeOH=50/1) to afford the title compound as a white foam (8.0 g,8.38 mmol, 66%).

Step 2

(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

The product from the previous step (40.0 g, 42.01 mmol) and H₂O (4.0 g,222 mmol, 4.0 mL, 5.3 eq) were added to a solution of Cl₂CHCOOH inCH₂Cl₂ (6% v/v, 400 mL) and the reaction mixture was stirred at RT for0.5 h, then washed with H₂O (4×200 mL). The organic layer was dried overanhydrous Na₂SO₄ and filtered. SiO₂ gel (80 g, previously treated withTEA) was combined with the filtrate, and the mixture was concentratedunder reduced pressure to obtain free flowing SiO₂ gel powder. Theresidue was purified by SiO₂ gel column chromatography (CH₂Cl₂/MeOH=50/1to 30/1) to give the title compound as a white solid (15.0 g, 23.08mmol, 55%);

MS(ES⁺) C₂₄H₃₄N₄O₇PSi requires: 549, found: 549.1 [M+H]⁺; ¹H-NMR (400MHz, 400 MHz, DMSO-d₆) δ ppm 11.15 (br s, 1H), 8.49-8.74 (m, 1H), 8.08(d, J=7.6 Hz, 2H), 7.78 (d, J=3.6 Hz, 1H), 7.60-7.67 (m, 1H), 7.51-7.57(m, 2H), 6.74 (d, J=3.6 Hz, 1H), 6.28 (d, J=6.2 Hz, 1H), 5.75 (s, 1H),4.56-4.74 (m, 2H), 4.16 (br s, 1H), 3.61-3.76 (m, 2H), 3.03 (q, J=7.2Hz, 5H), 1.19 (t, J=7.4 Hz, 7H), 0.69 (s, 9H), −0.09 (s, 3H), −0.27 (s,3H); ³¹P NMR (162 MHz, DMSO-d₆) δ ppm 0.72.

INTERMEDIATE C(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

Step 1

N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide

To a solution ofN-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-benzamide (35 g, 93.7 mmol) inpyridine (180 mL) was added DMTrCl (38.12 g, 112.5 mmol, 1.2 eq) and theresulting mixture was stirred at RT for 16 h. The mixture was thendiluted with CH₂Cl₂ (800 mL), washed with sat NaHCO₃ (2×400 mL) andbrine (400 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified by SiO₂ gel chromatography (petroleum ether/EtOAc=10/1 to 1/4)to give the title compound as a white solid (53.0 g, 78.4 mmol, 84%).

¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.26 (br s, 1H), 8.74 (s, 1H), 8.62 (s,1H), 8.05 (d, J=7.4 Hz, 2H), 7.60-7.72 (m, 1H), 7.48-7.58 (m, 2H), 7.32(d, J=7.2 Hz, 2H), 7.14-7.24 (m, 7H), 6.80 (dd, J=6.2, 8.7 Hz, 4H), 6.43(d, J=20.0 Hz, 1H), 5.73-5.85 (m, 1H), 5.61 (d, J=4.4 Hz, 1H), 4.76-4.99(m, 1H), 4.14 (br d, J=5.4 Hz, 1H), 3.64-3.79 (m, 7H), 3.19-3.33 (m,2H).

Step 2

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate

Phosphorous acid (18.2 g, 222 mmol, 15 eq) was co-evaporated three timeswith anhydrous pyridine (15 mL) and then dissolved with heating inanhydrous pyridine (150 mL). The mixture was allowed to cool to RT. Theproduct from the previous step (10 g, 14.8 mmol) was added, and theresulting mixture was cooled to 0° C. Pivaloyl chloride (17.85 g, 148mmol, 10 eq) was slowly added at 0° C. and the resulting mixture wasallowed to warm to RT and stirred for 16 h. The reaction mixture wasthen quenched with 1 M aq. TEAB (150 mL) and extracted with EtOAc (3×900mL). The combined organic layers were washed with 0.5 M aq. TEAB (900mL), brine (900 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by SiO₂gel chromatography (CH₂Cl₂/MeOH=50/1 to 20/1; 1% TEA) to give the titlecompound as a white foam (38 g).

Step 3

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

The product from the previous step (38 g, 45.19 mmol) and H₂O (4.0 g,222 mmol, 4.0 mL, 5 eq) were added to a solution of Cl₂CHCOOH in CH₂Cl₂(6% v/v, 380 mL) and the reaction mixture was stirred at RT for 0.5 h.The reaction mixture was filtered to give a red solid, which was washedwith CH₂Cl₂ (2×20 mL) to give the title compound as a white solid (15 g,30.87 mmol, 68%).

¹H-NMR (400 MHz, DMSO-d₆) δ ppm 11.24 (br s, 1H), 8.78 (s, 1H), 8.73 (s,1H), 8.02-8.08 (m, 2H), 7.76 (d, J=1.2 Hz, 0.5H), 7.62-7.68 (m, 1H),7.53-7.59 (m, 2H), 6.69 (s, 1H), 6.46 (dd, J=3.2, 16.6 Hz, 1H), 6.07 (d,J=1.4 Hz, 0.5H), 5.87-5.91 (m, 1H), 5.73-5.78 (m, 1H), 5.17-5.28 (m,1H), 4.22-4.28 (m, 1H), 3.64-3.84 (m, 2H).

INTERMEDIATE D(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite

Step 1

N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide

To a solution ofN-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-benzamide(100 g, 269.3 mmol) in pyridine (500 mL) at 0° C. were added DMAP (1.64g, 13.46 mmol, 0.05 eq) and DMTrCl (100.4 g, 296.2 mmol, 1.1 eq). Thereaction mixture was stirred at RT for 16 h, then quenched by additionof MeOH (500 mL). The volatiles were removed under reduced pressure andthe residue was purified by SiO₂ gel chromatography (1/1 petroleumether/EtOAc to 100% EtOAc) to give the title compound as a white foam(150 g, 223 mmol, 83%).

Step 2

N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(D-2a) andN-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyl-dimethylsilyloxy)-3-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(D-2b)

To a solution of the product from the previous step (200 g, 296.9 mmol)in THF (800 mL) and pyridine (15 mL) were added AgNO₃ (83.2 g, 489.8mmol, 82 mL, 1.65 eq) and TBSCl (78.7 g, 522.5 mmol, 1.76 eq), and themixture was stirred at RT for 16 h. The reaction mixture was filtered,diluted with H₂O (1.0 L) and extracted with EtOAc (3×1.0 L). Thecombined organic layers were washed with brine (2×1.0 L), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by SiO₂ gel chromatography (petroleum ether/EtOAc=3/1 to1/1) to give D-2a (114 g, 145 mmol, 49%) and D-2b (53 g, 68 mmol, 23%).

Step 3

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite

To a solution of D-2b (19.0 g, 24.1 mmol) in MeCN (200 mL) at 0° C. wereadded 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (7.99 g,26.5 mmol, 1.1 eq) and DCI (3.42 g, 28.9 mmol, 1.2 eq), and theresulting mixture was stirred for 3 h at RT under N₂ atmosphere. Thevolatiles were removed under reduced pressure and the residue waspurified by SiO₂ gel chromatography (petroleum ether/EtOAc=4/1 to 1.5/1;1% TEA) to give the title compound as a white foam (20.5 g, 20.74 mmol,86%).

MS(ES⁺) C₅₃H₆₇N₇O₈PSi requires: 988, found: 987.8 [M+H]⁺; ¹H-NMR (400MHz, CD₃CN) δ ppm 8.57 (s, 1H), 8.26 (s, 1H), 7.91 (br d, J=7.6 Hz, 2H),7.57 (m, 1H), 7.47-7.49 (m, 2H), 7.17-7.21 (m, 9H), 6.73-6.76 (m, 4H),6.10-6.19 (m, 1H), 5.10-5.14 (m, 1H), 4.64-4.68 (m, 1H), 4.10-4.14 (m,1H), 3.25-3.75 (m, 7H), 3.40-3.51 (m, 4H), 3.15-3.18 (m, 1H), 2.58 (t,J=6.0 Hz, 1H), 2.41 (t, J=6.0 Hz, 1H), 1.03-1.06 (m, 9H), 0.76-0.86 (m,12H), 0.00-0.93 (m, 6H); ³¹P NMR (162 MHz, CD₃CN) δ ppm 150.32, 149.58.

INTERMEDIATE E(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate triethylammonium salt

Step 1

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl) hydrogen phosphonate

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)-oxy)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (25 g, 25.3 mmol) in MeCN (12mL) was added H₂O (912 mg, 50.6 mmol, 2.0 eq) and pyridiniumtrifluoroacetate (5.86 g, 30.36 mmol, 1.2 eq) and the resulting mixturewas stirred for 5 minutes at RT. tert-Butylamine (1.85 g, 25.3 mmol, 1.0eq) was added and the resulting mixture was stirred for further 25minutes at RT and then concentrated under reduced pressure to give thetitle compound (23.4 g) as a white foam, which was used in the next stepwithout further purification.

Step 2

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

The crude product from the previous step (23.4 g, 25.3 mmol) was addedto a solution of Cl₂CHCOOH in CH₂Cl₂ (6% v/v, 200 mL). H₂O (2.28 g, 126mmol, 5.0 eq) was added and the resulting mixture was stirred for 20minutes at RT. The reaction mixture was quenched by the addition ofpyridine (30 mL) at RT, and was then concentrated under reducedpressure. The residue was purified by SiO₂ gel chromatography(CH₂Cl₂/MeOH=20/1 to 5/1) to give the title compound (15 g, 24.1 mmol).

Step 3

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate triethylammonium salt

The product from the previous step was dissolved in MeOH (100 mL), TEAresin (50.0 g; prepared from DOWEX® 50WX2-H⁺ by washing with deionizedwater until pH=7; then with 1N aq. TEA; then again with deionized wateruntil pH=7; then MeOH) was added and the mixture was stirred at RT for0.5 h. The reaction mixture was filtered, the filtrate was concentratedunder reduced pressure to give the title compound as a TEA salt (yellowsolid; 15 g, 23 mmol, 95%).

¹H-NMR (400 MHz, DMSO-d₆) δ ppm 8.57 (s, 1H), 8.26 (s, 1H), 7.91 (br d,J=7.6 Hz, 2H), 7.57 (m, 1H), 7.47-7.49 (m, 2H), 7.17-7.21 (m, 9H),6.73-6.76 (m, 4H), 6.10-6.19 (m, 1H), 5.10-5.14 (m, 1H), 4.64-4.68 (m,1H), 4.10-4.14 (m, 1H), 3.25-3.75 (m, 7H), 3.40-3.51 (m, 4H), 3.15-3.18(m, 1H), 2.58 (t, J=6.0 Hz, 1H), 2.41 (t, J=6.0 Hz, 1H), 1.03-1.06 (m,9H), 0.76-0.86 (m, 12H), 0.00-0.93 (m, 6H).

INTERMEDIATE F(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-chloro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate

Step 1

A mixture of(2R,3S,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yltrifluoromethanesulfonate (69.0 g, 0.11 mol, 1.0 eq) and LiCl (51.2 g,1.21 mol, 11.0 eq) in DMF (600 mL) was heated at 50° C. for 1.5 h. Thereaction mixture was diluted with DCM (500 mL) and washed with H₂O (300mL×3). The organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by SiO₂gel chromatography (PE:EtOAc=1:1) to give the title compound (46.0 g,89.5 mmol, 81.4% yield) as a yellow solid.

¹H NMR (400 MHz CDCl₃) δ ppm 8.30-8.38 (m, 1H), 8.06 (s, 1H), 6.20 (d,J=5.9 Hz, 1H), 5.82-5.95 (m, 2H), 4.96-5.04 (m, 1H), 4.56-4.63 (m, 1H),4.17 (q, J=3.5 Hz, 1H), 3.96 (dd, J=11.4, 4.3 Hz, 1H), 3.77 (dd, J=11.4,3.0 Hz, 1H), 0.92 (d, J=13.9 Hz, 18H), 0.14 (d, J=8.8 Hz, 6H), 0.08 (s,6H).

Step 2.

To a solution of the product from previous step (46.0 g, 89.4 mmol, 1.0eq) in Py (460 mL) at 0° C. was added BzCl (25.2 g, 179 mmol, 20.5 mL,2.0 eq) dropwise under N₂, and the resulting mixture was stirred at 25°C. for 16 hrs. The reaction was diluted with H₂O (200 mL) and extractedwith EtOAc (200 mL×3). The combined organic layers were dried overNa₂SO₄, filtered and concentrated under reduced pressure to giveN-benzoyl-N-(9-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chlorotetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(60.0 g) as yellow oil which was used for the next step without furtherpurification. MS(ES⁺) C₃₆H₄₈ClN₅O₅Si₂ requires: 721, found: 722 [M+H]⁺;

Step 3

To a solution of the product from previous step (60.0 g, 97 mmol, 1.0eq) in THF (600 mL) was added TBAF (1 M in THF, 290 mL, 3.0 eq). Thenthe mixture was stirred at 25° C. for 16 hrs. The volatiles were removedunder reduced pressure to giveN-benzoyl-N-(9-((2R,3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(48.0 g) as a yellow oil which was used for the next step withoutfurther purification. MS(ES⁺) C₂₄H₂₀ClN₅O₅ requires: 493, found: 494[M+H]⁺;

Step 4

To a solution of the product from previous step (48.0 g, 97.2 mmol, 1.0eq) in THF (500 mL) was added NH₃.H₂O (28% in water, 8.0 mL, 7.28 g,54.0 mmol, 0.5 eq). The resulting mixture was stirred at 25° C. for 1hr, during which time a solid product was formed. The suspension wasfiltered and the filter cake was washed with THF (300 mL×3). Thefiltrate was concentrated under reduced pressure to giveN-(9-((2R,3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(36 g, 92.3 mmol, 95% yield) as a white solid product; MS(ES⁺)C₁₇H₁₆ClN₅O₄ requires: 389, found: 390 [M+H]⁺;

Steps 5 to 7

The transformations depicted in the above scheme were performedaccording to the procedures previously described for the synthesis ofIntermediate C, steps 1 to 3.

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-chloro-2(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (Intermediate F)

white solid (13.0 g, 27.8 mmol, 30% over three steps); ¹H NMR: (400 MHzDMSO-d₆) δ ppm 11.28 (br s, 1H), 8.78 (br d, J=4.0 Hz, 2H), 8.05 (br d,J=7.4 Hz, 2H), 7.60-7.68 (m, 1H), 7.55 (br t, J=7.5 Hz, 2H), 6.34 (br d,J=5.9 Hz, 1H), 5.27 (br d, J=5.1 Hz, 1H), 5.02 (br s, 1H), 4.30 (br s,1H), 3.74 (br s, 2H), 3.01-3.11 (m, 2H); ³¹P NMR: (162 MHz DMSO-d₆) δ0.63 ppm.

EXAMPLE 1a AND EXAMPLE 1b Cyclic dinucleotides RR-CD-A-7dA andSR-CD-A-7dA dithio-[R_(p),R_(p)]-cyclic-[A(2′,5′)p-7dA(3′,5′)p]dithio-[S_(p),R_(p)]-cyclic-[A(2′,5′)p-7dA (3′,5′)p]

Step 1

Compound 1-1

To a solution of Intermediate B (4.00 g, 6.16 mmol) in CH₃CN (50 mL) wasadded pyridine-TFA (2.38 g, 12.3 mmol, 2.0 eq) followed by a mixture ofIntermediate D (6.70 g, 6.78 mmol, 1.10 eq) and 3 Å molecular sieves(1.0 g, 24.6 mmol, 4.0 eq) in CH₃CN (50 mL), and the resulting mixturewas stirred for 30 minutes at RT. DDTT (1.52 g, 7.39 mmol, 1.20 eq) wasthen added and the mixture was stirred at RT for further 30 minutes. Thevolatiles were removed under reduced pressure to afford the crudecompound 1-1 (9.04 g), which was used without further purification inthe next step.

Step 2

Compound 1-2

To a solution of Cl₂CHCOOH in CH₂Cl₂ (6% v/v, 200 mL) was added H₂O (2.0g, 111 mmol, 2.0 mL, 18.0 eq) and compound 1-1 from the previous step(9.04 g, assume 6.16 mmol). The reaction mixture was stirred at RT for0.5 h, then quenched with pyridine (120 mL) and concentrated underreduced pressure, to afford crude compound 1-2 (7.18 g), which was usedwithout further purification in the next step.

MS(ES⁺) C₅₀H₆₇N₁₀O₁₃P₂SSi₂ requires: 1165, found: 1165.3 [M+H]⁺.

Step 3

Compound 1-3

To a solution of compound 1-2 from the previous step (7.18 g, assume6.16 mmol) in pyridine (50 mL) was added DMOCP (3.98 g, 21.6 mmol, 3.5eq). The mixture was stirred for 0.5 h at RT. To the mixture was thenadded Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 1.85 g,9.24 mmol, 1.5 eq), and the resulting mixture and stirred at RT forfurther 30 minutes. The reaction mixture was quenched by addition of3.4% aq. NaHCO₃ (1.0 L), and then extracted with EtOAc (2×500 mL). Thecombined organic layers were washed with brine (300 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by SiO₂ gel chromatography (CH₂Cl₂/MeOH=30/1 to15/1) to give compound 1-3 (3.0 g, 2.04 mmol) as a mixture ofdiastereoisomers which was used as such in the following step.

Step 4

Compound 1-4

To a solution of compound 1-3 from the previous step (3.0 g, 2.04 mmol)in MeOH (30 mL) was added NH₄OH (32.8 g, 935 mmol, 458 eq). The mixturewas stirred at 50° C. for 12 h in a pressure safe steel vessel, thenconcentrated under reduced pressure. The residue was purified byprep-HPLC (PHENOMENEX® LUNA® C18 250*50 10 um; mobile phase: A: H₂O (10mM NH₄HCO₃); B: MeCN; A %-B %=20%-50%, 20 minutes) to give two products:compound 1-4a (R_(p)R_(p) or S_(p)R_(p) diastereoisomer; 380 mg, 0.391mmol;) and compound 1-4b (S_(p)R_(p) or R_(p)R_(p) diastereoisomer; 350mg, 0.349 mmol) as a white solids.

Step 5

Compound 1-5

To a solution of compound 1-4a (200 mg, 218 umol) in MeOH (5.0 mL) wasadded NH₄F (80.7 mg, 2.18 mmol, 10.0 eq) and the resulting mixture wasstirred at 60° C. for 16 h. The volatiles were removed under reducedpressure and the residue was purified by prep-HPLC [Waters Xbridge150*25 5 um; mobile phase: A: H₂O (10 mM NH₄HCO₃); B: MeCN; A %-B%=1%-20%, 10.5 minutes] to give compound 1-5a (30 mg, 40 umol) as awhite solid.

Reaction of compound 1-4b in a similar manner afforded compound 1-5b (30mg, 40 umol) as a white solid.

Step 6

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-17-(6-amino-9H-purin-9-yl)-8-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-9,18-dihydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione (1-5),disodium salt (Example 1a and Example 1b)

To a solution of compound 1-5a (30.0 mg, 41.5 umol) in H₂O (5.0 mL) wasadded DOWEX®-50WX8 (Na⁺ form; 300 mg) and the mixture was stirred at RTfor 0.5 h. The reaction was then filtered, and the filtrate waslyophilized to give Example 1a (R_(p), R_(p) or S_(p), R_(p) 28.0 mg,38.1 umol) as a white solid.

MS(ES⁺) C₂₁H₂₆N₉O₁₀P₂S₂ requires: 690, found: 690.0 [M+H]⁺; ¹H-NMR (400MHz, DMSO-d₆) δ ppm 8.52 (s, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 7.50 (d,J=3.6 Hz 1H), 6.85 (d, J=3.6 Hz, 1H), 6.09-6.13 (m, 2H), 5.46 (d, J=8.8Hz, 1H), 5.28-5.30 (m, 1H) 4.63 (d, J=4 Hz, 1H), 4.03-4.27 (m, 5H),3.64-3.69 (m, 2H); ³¹P NMR (162 MHz, DMSO-d₆) δ ppm 60.19, 56.77;R_(t)=1.797 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobilephase: A: H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2 minutes]

Reaction of compound 1-5b in a similar manner gave Example 1b (S_(p),R_(p) or R_(p), R_(p) 30.0 mg, 39.6 umol) as a white solid.

MS(ES⁺) C₂₁H₂₆N₉O₁₀P₂S₂ requires: 690, found: 690.0 [M+H]⁺; ¹H-NMR (400MHz, DMSO-d₆) δ ppm 8.50 (s, 1H), 8.29 (s, 1H), 8.19 (s, 1H), 7.62 (d,J=3.2 Hz 1H), 6.85 (d, J=4.0 Hz, 1H), 6.09-6.14 (m, 2H), 5.19-5.29 (m,2H), 4.68 (dd, J=7.6 Hz, 1H), 4.37 (d, J=4.0 Hz, 1H), 4.11-4.20 (m, 3H),3.95-3.99 (m, 1H), 3.69-3.81 (m, 2H); ³¹P NMR (162 MHz, CD₃OD) δ ppm59.29, 51.96; R_(t)=2.101 minutes [Waters XBridge Shield RP18 2.1*50 mm,5 um; mobile phase: A: H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2minutes].

EXAMPLE 2a AND EXAMPLE 2b Cyclic dinucleotides RR-CD-A-2′F-A andSR-CD-A-2′F-A; dithio-[R_(p),R_(p)]-cyclic-[A(2′,5′)p-2′F-A(3′,5′)p]dithio-[S_(p),R_(p)]-cyclic-[A(2′,5′)p-2′F-A (3′,5′)p]

Step 1

Compound 2-1

To a solution of Intermediate C (4.0 g, 9.15 mmol) in CH₂Cl₂ (40 ml) wasadded TEA (463 mg, 4.58 mmol, 0.50 eq). The mixture was stirred for 5minutes at RT and the volatiles were removed under reduced pressure. Theresidue was dissolved in CH₃CN (40.00 mL) and pyridine-TFA (3.53 g, 18.3mmol, 2.0 eq) was added, followed by a mixture of Intermediate D (9.04g, 9.15 mmol, 1.0 eq) and 3 Å molecular sieves (1.48 g, 36.6 mmol, 4.0eq) in CH₃CN (40 mL), and the resulting mixture was stirred for 30minutes at RT. DDTT (2.25 g, 10.98 mmol, 1.2 eq) was added and themixture was stirred at RT for further 30 minutes. The volatiles wereremoved under reduced pressure to afford the crude compound 2-1 (12.4g), which was used without further purification in the next step.

Step 2

Compound 2-2

To a solution of Cl₂CHCOOH in CH₂Cl₂ (6% v/v, 200 mL) was added compound2-1 from the previous step (12.4 g, assume 9.15 mmol) and H₂O (2.00 g,111 mmol, 2.0 mL, 12.1 eq). The reaction mixture was stirred at RT for0.5 h, then quenched with pyridine (100 mL) and concentrated underreduced pressure to afford compound 2-2 (9.64 g), which was used withoutfurther purification in the next step.

Step 3

Compound 2-3

To a solution of compound 2-2 from the previous step (9.63 g, assume9.15 mmol) in pyridine (200 mL) was added DMOCP (5.90 g, 32.0 mmol, 3.5eq) and the mixture was stirred for 0.5 h at RT.3H-1,2-Benzodithiol-3-one 1,1-dioxide (2.75 g, 13.7 mmol, 1.5 eq) wasthen added, and the resulting mixture and stirred at RT for further 30minutes. The reaction mixture was quenched by addition of 3.4% aq.NaHCO₃ (1.0 L), and then extracted with EtOAc mL (2×500 mL). Thecombined organic layers were washed with brine (300 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by SiO₂ gel chromatography (CH₂Cl₂/MeOH=30/1 to10/1) to give compound 2-3 (3.2 g, 2.1 mmol) as a mixture ofdiastereoisomers which was used as such in the following step.

Step 4

Compound 2-4

To a solution of compound 2-3 from the previous step (2.0 g, 1.87 mmol)in MeOH (10 mL) was added NH₄OH (18.2 g, 519 mmol, 277 eq). The mixturewas stirred at 50° C. for 16 h in a pressure safe steel vessel, thenconcentrated under reduced pressure. The residue was purified byprep-HPLC [PHENOMENEX® LUNA® C18 250*50 10 um; mobile phase: A: H₂O (10mM NH₄HCO₃); B: MeCN; A %-B %=10%-40%, 20 minutes] to give two products:compound 2-4a (R_(p)R_(p) or S_(p)R_(p) diastereoisomer, 180 mg, 0.209mmol) and compound 2-4b (S_(p)R_(p) or R_(p)R_(p) diastereoisomer, 200mg, 0.228 mmol) as white solids.

Step 5

Compound 2-5

To a solution of compound 2-4a (100 mg, 119 umol) in MeOH (3.0 mL) wasadded NH₄F (44.1 mg, 1.19 mmol, 10 eq) and the resulting mixture wasstirred at 60° C. for 16 h. The reaction mixture was then allowed tocool to RT and concentrated under reduced pressure. The residue wastaken up in H₂O (0.5 mL), cooled to 10° C. and kept stirring for 30minutes, then filtered and the filter cake was collected to givecompound 2-5a (30.0 mg, 41.3 umol) as a white solid.

MS(ES⁺) C₂₀H₂₄FN₁₀O₉P₂S₂ requires: 693, found: 693.2 [M+H]⁺; ¹H-NMR (400MHz, CD₃OD) δ ppm 8.98 (s, 1H), 8.22 (s, 2H), 7.82 (s, 1H), 6.45 (d,J=14.4 Hz, 1H), 6.33 (d, J=8.0 Hz, 1H), 5.62 (d, J=53.8 Hz, 1H), 5.32(m, 1H), 5.07-5.13 (m, 1H), 4.36-4.46 (m, 5H), 4.06 (d, J=11.2 Hz, 1H),3.86-3.90 (m, 1H).

Reaction of compound 2-4b in a similar manner afforded compound 2-5b(30.0 mg, 41.3 umol) as a white solid.

MS(ES⁺) C₂₀H₂₄FN₁₀O₉P₂S₂ requires: 693, found: 693.2[M+H]⁺; ¹H-NMR (400MHz, CD₃OD) δ ppm 8.76 (s, 1H), 8.49 (s, 1H), 8.24 (s, 1H), 8.18 (s,1H), 6.34-6.41 (m, 2H), 5.70 (dd, J=51.8 Hz, 1H), 5.22-5.239 (m, 2H),4.50-4.59 (m, 4H), 4.32 (s, 1H), 4.03-4.07 (m, 1H).

Step 6

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-8,17-di(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo-[13.2.1.0^(6,10)]octadecane-3,12-dione (2-5),disodium salt (Example 2a and Example 2b)

To a solution of compound 2-5a (30.0 mg, 41.3 umol) in H₂O (10.0 mL) wasadded Dowex®-50WX8 (Na⁺ form; 300 mg) and the mixture was stirred at RTfor 4 h. The reaction was then filtered, and the filtrate waslyophilized to give Example 2a (R_(p)R_(p) or S_(p)R_(p); 30.0 mg, 40.6umol) as a white solid.

MS(ES⁺) C₂₀H₂₄FN₁₀O₉P₂S₂ requires: 693, found: 693.0 [M+H]⁺; ¹H-NMR (400MHz, DMSO-d₆) δ ppm 8.56 (s, 1H), 8.40 (s, 1H), 8.17 (s, 1H), 8.13 (s,1H), 7.34 (s, 2H), 7.22 (s, 2H), 6.23 (m, 1H), 6.09 (d, J=8.4 Hz 1H),5.71 (d, J=52.8 Hz, 1H), 5.54 (s, 1H), 5.15-5.30 (m, 2H), 3.91-4.37 (m,5H), 3.67-3.70 (m, 1H); ³¹P NMR (162 MHz, CD₃OD) δ ppm 55.97, 53.66;R_(t)=1.384 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobilephase: A: H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Reaction of compound 2-5b in a similar manner gave Example 2b(S_(p)R_(p) or R_(p)R_(p), 28.0 mg, 40.6 umol) as a white solid.

MS(ES⁺) C₂₀H₂₄FN₁₀O₉P₂S₂ requires: 693, found: 693.0 [M+H]⁺; ¹H-NMR (400MHz, CD₃OD) δ ppm 8.86 (br s, 1H), 8.39 (s, 1H), 8.19 (s, 1H), 8.02 (brs, 1H), 6.35-6.41 (m, 2H), 5.70 (d, J=51.8 Hz, 1H), 5.22-5.27 (m, 2H),4.35-4.60 (m, 5H), 4.05-4.08 (m, 2H); ³¹P NMR (162 MHz, CD₃OD) δ ppm57.39, 52.28; R_(t)=1.644 minutes [Waters XBridge Shield RP18 2.1*50 mm,5 um; mobile phase: A: H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2minutes].

EXAMPLE 3a AND EXAMPLE 3b Cyclic dinucleotides RR-CD-7dA-A andSR-CD-7dA-A dithio-[R_(p),R_(p)]-cyclic-[7dA(2′,5′)p-A(3′,5′)p]dithio-[S_(p),R_(p)]-cyclic-[7dA(2′,5′)p-A (3′,5′)p]

Step 1

Compound 3-1

To a solution of Intermediate E (3.0 g, 4.61 mmol) in CH₃CN (30 mL) wasadded pyridine-TFA (1.78 g, 9.22 mmol, 2.0 eq) followed by a mixture ofIntermediate A (5.0 g, 5.07 mmol, 1.1 eq) and molecular sieves (0.8 g,18.4 mmol, 4.00 eq) in CH₃CN (30 mL), and the resulting mixture wasstirred for 30 minutes at RT. DDTT (1.14 g, 5.53 mmol, 1.2 eq) was thenadded and the mixture was stirred at RT for further 30 minutes. Thevolatiles were removed under reduced pressure to afford crude compound3-1 (6.77 g), which was used without further purification in the nextstep.

Step 2

Compound 3-2

To a solution of Cl₂CHCOOH acid in CH₂Cl₂ (6% v/v, 100 mL) was added H₂O(83.1 mg, 4.61 mmol, 83.12 uL, 1.00 eq) and compound 3-1 from theprevious step (6.77 g, assume 4.61 mmol, 1.00 eq). The reaction mixturewas stirred at RT for 0.5 h, then quenched with pyridine (80 mL) andconcentrated under reduced pressure to afford compound 3-2 (5.48 g),which was used without further purification in the next step.

Step 3

Compound 3-3

To a solution of compound 3-2 from the previous step (5.48 g, assume4.61 mmols) in pyridine (150 mL) was added DMOCP (4.77 g, 25.8 mmol, 5.5eq) and the mixture was stirred for 0.5 h at RT.3H-1,2-Benzodithiol-3-one 1,1-dioxide (1.41 g, 7.05 mmol, 1.5 eq) wasthen added, and the resulting mixture and stirred at RT for further 30minutes. The reaction mixture was quenched by addition of 3.4% aq.NaHCO₃ (600 mL), and then extracted with EtOAc (2×300 mL). The combinedorganic layers were washed with brine (200 mL), dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by SiO₂ gel chromatography (CH₂Cl₂/MeOH=30/1 to 15/1) togive compound 3-3 (3.0 g, 1.53 mmol) as a mixture of diastereoisomerswhich was used as such in the following step.

Step 4

Compound 3-4

To a solution of compound 3-3 from the previous step (3.0 g, 1.53 mmol)in MeOH (30 mL) was added NH₄OH (16.4 g, 468 mmol, 307 eq). The mixturewas stirred at 50° C. for 16 h in a pressure safe steel vessel, thenconcentrated under reduced pressure. The residue was purified byprep-HPLC [PHENOMENEX® LUNA® C18 250*50 10 um; mobile phase: A: H₂O (10mM NH₄HCO₃); B: MeCN; A %-B %=20%-45%, 20 minutes] to give compound 3-4a(R_(p)R_(p) or S_(p)R_(p) diastereoisomer, 220 mg, 208 umol) andcompound 3-4b (S_(p)R_(p) or R_(p)R_(p) diastereoisomer, 220 mg, 208umol) as white solids.

Step 5

Compound 3-5

To a solution of compound 3-4a (100 mg, 105 umol) in MeOH (3.0 mL) wasadded NH₄F (38.9 mg, 1.05 mmol, 10 eq) and the resulting mixture wasstirred at 60° C. for 12 h. The reaction mixture was then allowed toreach RT and concentrated under reduced pressure. The residue was takenup in H₂O (1 mL) at 40° C., cooled to 5° C. and kept stirring for 30minutes, then filtered and the filter cake was collected to givecompound 3-5a (20.0 mg, 26.3 umol,) as a white solid.

Reaction of compound 3-4b in a similar manner afforded compound 3-5b(33.0 mg, 45.6 umol) as a white solid.

Step 6

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-17-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0^(6,10)]octadecane-3,12-dione, disodiumsalt (Example 3a and Example 3b)

To a solution of compound 3-5a (20.0 mg, 27.6 umol) in H₂O (5 mL) wasadded DOWEX® 50WX8 (Na⁺ form; 300 mg) and the mixture was stirred at RTfor 3 h. The reaction was then filtered, and the filtrate waslyophilized to give Example 3a as a white solid (R_(p)R_(p) orS_(p)R_(p), 18.0 mg, 24.5 umol).

MS(ES⁺) C₂₁H₂₆N₉O₁₀P₂S₂ requires: 690, found: 690.0 [M+H]⁺; ¹H-NMR (400MHz, CD₃OD) δ ppm 8.33 (s, 1H), 8.20 (s, 1H), 8.05 (s, 1H), 7.84 (d,J=4.0 Hz 1H), 6.62 (d, J=3.6 Hz, 1H), 6.51 (d, J=8.4 Hz, 1H), 6.12 (d,J=6.8 Hz, 1H), 5.59-5.60 (m, 1H), 5.35-5.59 (m, 1H), 5.21-5.24 (m, 1H),4.25-4.45 (m, 4H), 3.99 (d, J=12.0 Hz, 1H), 3.90-3.92 (m, 1H); ³¹P NMR(162 MHz, CD₃OD) δ ppm 60.36, 60.28; R_(t)=1.497 minutes [Waters XBridgeShield RP18 2.1*50 mm, 5 um; mobile phase: A: H₂O+10 mM NH₄HCO₃; B:MeCN; A %-B %=0%-30%, 5.2 minutes].

Reaction of compound 3-5b in a similar manner gave Example 3b(S_(p)R_(p) or R_(p)R_(p), 33.0 mg, 44.9 umol) as a white solid.

MS(ES⁺) C₂₁H₂₆N₉O₁₀P₂S₂ requires: 690, found: 690.0 [M+H]⁺; ¹H-NMR (400MHz, CD₃OD) δ ppm 8.34 (s, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 7.97 (d,J=3.6 Hz 1H), 6.72 (d, J=4 Hz, 1H), 6.56 (d, J=8.0 Hz, 1H), 6.12 (d,J=3.2 Hz, 1H), 5.20-5.31 (m, 2H), 4.98-5.01 (m, 1H), 4.71 (d, J=4 Hz,1H), 4.33-4.49 (m, 1H), 4.25-4.30 (m, 3H), 4.07-4.12 (m, 1H); ³¹P NMR(162 MHz, CD₃OD) δ ppm 57.30, 53.90; R_(t)=1.647 minutes [Waters XBridgeShield RP18 2.1*50 mm, 5 um; mobile phase: A: H₂O+10 mM NH₄HCO₃; B:MeCN; A %-B %=0%-30%, 5.2 minutes].

EXAMPLE 4a, 4b, 4c AND 4b Cyclic dinucleotides RR-CD-A-2′Cl-A,RS-CD-A-2′Cl-A, SS-CD-A-2′Cl-A, and SR-CD-A-2′ Cl-A; dithio-[R_(p),R_(p)]-cyclic-[A(2′,5′)p-2′Cl-A(3′,5′)p], dithio-[R_(p),S_(p)]-cyclic-[A(2′,5′)p-2′Cl-A(3′,5′)p], dithio-[S_(p),S_(p)]-cyclic-[A(2′,5′)p-2′Cl-A(3′,5′)p]dithio-[S_(p),R_(p)]-cyclic-[A(2′,5′)p-2′Cl-A (3′,5′)p]

Step 1

Compound 4-1.

To a solution of Int F (3.00 g, 6.61 mmol, 1.0 eq) in DCM (60 mL) wasadded TEA (0.334 g, 3.31 mmol, 0.46 uL, 0.5 eq). The volatiles wereremoved under reduced pressure, pyridine-TFA (2.55 g, 13.2 mmol, 2.0 eq)was added to the residue, and the mixture was co-evaporated three timeswith anhydrous CH₃CN (40 mL). The residue was dissolved in anhydrousCH₃CN (30 mL) and stirred with 3 Å molecular sieves (3.00 g, 6.61 mmol,1.0 eq) for five minutes. In a separate vessel Int D (6.53 g, 6.61 mmol,1.0 eq) was co-evaporated three times with anhydrous CH₃CN (20 mL), thendissolved in anhydrous CH₃CN (30 mL). The resulting solution of Int Dwas added to the mixture of Int F, pyridine-TFA and 3 Å molecularsieves, followed by DDTT (1.63 g, 7.93 mmol, 1.2 eq), and the resultingmixture was stirred at 25° C. for 30 min. The mixture was thenconcentrated under reduced pressure to give Compound 4-1 (9.0 g) as ayellow solid which was used for the next step without furtherpurification.

Step 2

Compound 4-2.

To a solution of Compound 4-1 (9.0 g, 6.56 mmol, 1.0 eq) in DCM (90 mL)was added Cl₂CHCOOH acid (2.7 mL). The mixture was stirred at 25° C. for1 hr, and triethylsilane (4.57 g, 39.3 mmol, 6.28 mL, 6.0 eq) was addedfollowed by pyridine (45.0 mL). The resulting mixture was stirred at RTfor 30 minutes, then concentrated under reduced pressure. The residuewas purified by SiO₂ gel chromatography (DCM:MeOH=10:1 to 3:1) to giveCompound 4-2 (2.12 g, 1.98 mmol, 30% yield) as a light yellow solid.MS(ES⁺) C₄₃H₅₀ClN₁₁O₁₂P₂SSi requires: 1069, found: 1070 [M+H]⁺.

Step 3

Compound 4-3a and Compound 4-3b.

Compound 4-2 (2.12 g, 1.98 mmol, 1.0 eq) was co-evaporated three timeswith anhydrous Pyridine (40.0 mL), dissolved in anhydrous Pyridine (40.0mL) and the solution was cooled to 0° C. DMOCP (1.28 g, 6.93 mmol, 3.5eq) was added, and the mixture was stirred for 0.5 h at RT.3H-1,2-Benzodithiol-3-one 1,1-dioxide (0.595 g, 2.97 mmol, 1.5 eq) wasthen added, and the resulting mixture and stirred at RT for further 30minutes. The reaction mixture was quenched by addition of 3.4% aq.NaHCO₃ (600 mL), and then extracted with EtOAc (2×300 mL). The combinedorganic layers were washed with brine (200 mL), dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by SiO₂ gel chromatography (CH₂Cl₂/MeOH=30/1 to 10/1) togive compound 4-3a (0.470 g, 0.348 mmol, 17% yield) and compound 4-3b(0.380 g, 0.28 mmol, 14% yield), each one as a mixture ofdiastereoisomers of undefined stereochemistry at the phosphorothioatecenters.

Step 4

Compound 4-4a, 4-4b, 4-4c, 4-4d.

To a solution of Compound 4-3a (0.47 g, 0.43 mmol, 1.0 eq) in MeOH (3mL) was added NH₄OH (4.2 g, 519 mmol, 277 eq). The mixture was stirredat 50° C. for 16 h in a pressure safe steel vessel, then cooled to RTand concentrated under reduced pressure. The residue was purified byprep-HPLC [Agela Durashell® C18 150×25 5 um; mobile phase: A: H₂O (10 mMNH₄HCO₃); B: MeCN; A %-B %=10%-40%, 10.5 minutes] to give twostereoisomer products of undefined stereochemistry at thephosphorothioate centers; compound 4-4a (93.0 mg, 0.11 mmol, 25% yield)and compound 4-4c (105 mg, 0.13 mmol, 29% yield), both as white solids.

Compound 4-3b (0.380 g, 0.28 mmol) was reacted in the same manner togive compounds 4-4b (82.0 mg, 99.6 umol, 28% yield) and 4-4d (82.0 mg,99.6 umol, 28% yield) as two stereoisomers of undefined stereochemistryat the phosphorothioate centers, both as white solids.Step 5

Compound 4-5a, 4-5b, 4-5c, 4-5d.

To a solution of compound 4-4a (93.0 mg, 0.11 mmol, 1.0 eq) in MeOH (3.0mL) was added NH₄F (80 mg, 2.17 mmol, 20.0 eq) and the resulting mixturewas stirred at 65° C. for 16 h. The reaction mixture was then allowed tocool to RT and concentrated under reduced pressure. The residue waspurified by prep-HPLC [Agela Durashell® C18 150×25 5 um; mobile phase:A: H₂O (10 mM NH₄HCO₃); B: MeCN; A %-B %=10%-40%, 10.5 minutes] to givecompound 4-5a (50.0 mg, 65.1 umol, 60% yield) as a white solid;Compounds 4-4b, 4-4c and 4-4d were reacted in the same manner to givethe following compounds: 4-5b (40.0 mg, 56.4 umol, 56% yield); 4-5c(58.0 mg, 78.1 umol, 64% yield); and 4-5d (20.0 mg, 22.6 umol, 23%yield).

Step 6

EXAMPLE 4a, 4b, 4c AND 4d

To a solution of compound 4-5a (50.0 mg, 67.3 umol, 1.0 eq) in H₂O (20.0mL) was added Dowex®-50WX8 (Na⁺ form; 500 mg) and the mixture wasstirred at RT for 4 h. The reaction was then filtered, and the filtratewas lyophilized to give Example 4a (36.5 mg, 46.6 umol, 69% yield) as awhite solid; single stereoisomer, undefined stereochemistry at thephosphorothioate centers; MS(ES⁺) C₂₀H₂₃ClN₁₀O₉P₂S₂ requires: 708,found: 709 [M+H]⁺; ¹H-NMR (400 MHz D₂O) δ ppm 8.68 (s, 1H), 8.13 (s,1H), 8.07 (s, 1H), 8.01 (s, 1H), 6.28 (d, J=2.0 Hz, 1H), 6.17 (d, J=8.4Hz, 1H), 5.29-5.40 (m, 2H), 5.18-5.26 (m, 1H), 4.62 (d, J=4.0 Hz, 1H),4.57 (br s, 1H), 4.42-4.51 (m, 2H), 4.14-4.25 (m, 2H), 4.09 (br d,J=12.8 Hz, 1H); ³¹P NMR (162 MHz, D₂O) δ ppm 55.69, 54.83; R_(t)=1.64minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A:H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Compounds 4-5b, 4-5c and 4-5d were reacted in the same manner to givethe following compounds, all as single stereoisomers, with undefinedstereochemistry at the phosphorothioate centers:

Example 4b (40.0 mg, 48.2 umol, 89.6% yield); MS(ES⁺) C₂₀H₂₃ClN₁₀O₉P₂S₂requires: 708, found: 709 [M+H]⁺; ¹H-NMR (400 MHz D₂O) δ ppm 8.42 (s,1H), 8.16 (s, 1H), 8.04 (s, 1H), 7.94 (s, 1H), 6.26 (d, J=3.1 Hz, 1H),6.20 (d, J=8.3 Hz, 1H), 5.33-5.43 (m, 2H), 5.09-5.13 (m, 1H), 4.55-4.61(m, 2H), 4.42-4.49 (m, 3H), 4.34-4.41 (m, 1H), 4.05-4.14 (m, 2H); ³¹PNMR (162 MHz, D₂O) δ ppm 56.58, 54.75; R_(t)=1.74 minutes [WatersXBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H₂O+10 mM NH₄HCO₃;B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Example 4c (35.2 mg, 46.6 umol, 59.7% yield), MS(ES⁺) C₂₀H₂₃ClN₁₀O₉P₂S₂requires: 708, found: 709 [M+H]⁺; ¹H-NMR (400 MHz D₂O) δ ppm 8.65 (s,1H), 8.19 (s, 1H), 8.08 (s, 1H), 8.05 (s, 1H), 6.37 (s, 1H), 6.17 (d,J=8.3 Hz, 1H), 5.42 (d, J=4.8 Hz, 1H), 5.33 (m, 1H), 5.08-5.16 (m, 1H),4.93 (d, J=3.9 Hz, 1H), 4.62 (br d, J=9.0 Hz, 1H), 4.44-4.51 (m, 2H),4.27 (m, 1H), 4.18 (br s, 2H); ³¹P NMR (162 MHz, D₂O) δ ppm 53.99,51.92; R_(t)=2.09 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um;mobile phase: A: H₂O+10 mM NH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2minutes].

Example 4d (17.0 mg, 24.0 umol, 89.1% yield), MS(ES⁺) C₂₀H₂₃ClN₁₀O₉P₂S₂requires: 708, found: 709 [M+H]⁺; ¹H-NMR (400 MHz D₂O) δ ppm 8.34 (s,1H), 8.10 (s, 1H), 8.04 (s, 1H), 7.86 (s, 1H), 6.30 (s, 1H), 6.14 (d,J=8.4 Hz, 1H), 5.36 (m, 1H), 5.24 (m, 1H), 5.05 (d, J=4.4 Hz, 1H), 4.80(d, J=4.0 Hz, 1H), 4.55 (br d, J=8.0 Hz, 1H), 4.36-4.44 (m, 2H), 4.32(ddd, J=12.0, 6.4, 2.0 Hz, 1H), 4.20 (m, 1H), 4.02 (dd, J=11.2, 3.6 Hz,1H); ³¹P NMR (162 MHz, D₂O) δ ppm 54.64, 52.01; R_(t)=2.03 minutes[Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H₂O+10 mMNH₄HCO₃; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

The following compounds, or a salt, ester, prodrug, or tautomer thereof,can generally be made using the methods described above. It is expectedthat these compounds when made will have activity similar to those thathave been prepared.

Biological Activity Assays

THP-1 Dual Assay

The INVIVOGEN® THP-1 Dual™ assay (catalog code: thpd-nfis) was used toevaluate the listed compounds as agonist of the STING receptor. TheTHP-1 Dual™ cells are derived from the human monocytic cell line THP-1by stable integration of two inducible reporter constructs. This assayenables simultaneous study of the two main signaling pathways for STING:(a) the NF-κB pathway, by monitoring the activity of secreted embryonicalkaline phosphatase (SEAP); and (b) the Interferon regulatory factor(IRF) pathway, by assessing the activity of a secreted luciferase(Lucia™).

The procedure as set forth by the manufacturer was followed, with thefollowing modifications: (1) plates are incubated 18 h after addition ofcell suspension, and (2) the optional differentiation step is notemployed.

Tables 1 and 2. Biological Activity

TABLE 1 Fold induction of IRF3 activation in THP-1 Dual ™ cells upontreatment with increasing concentrations of STING agonist. 0.1 0.5 1 510 mg/mL mg/mL mg/mL mg/mL mg/mL 3′,5′-c-di-GMP 1.07 1.26 1.32 2.87 5.93ML-RS-CDA* 1.31 11.55 56.72 144.86 141.76 ML-RR-CDA** 2.65 4.57 8.91148.89 166.88 Example 1a 1.14 1.84 9.77 112.64 157.93 Example 1b 0.991.88 3.52 25.91 87.63 Example 2a 27.89 138.29 148.06 147.12 143.02Example 2b 16.46 95.25 156.38 159.53 157.10 Example 3a 1.41 0.98 2.073.04 4.29 Example 3b 1.38 1.48 1.56 5.05 6.70 Example 4d 1.99 nd 5.89 nd22.37 *= dithio-[R_(p),S_(p)]-cyclic-[A(2′,5′)p-A(3′,5′)p]; **=dithio-[R_(p),R_(p)]-cyclic-[A(2′,5′)p-A(3′,5′)p]; prep'd as in WO2014/189805. Examples 4a-4c did not show significant activity.

TABLE 2 Fold induction of NF-κB activation THP-1 Dual ™ cells upontreatment with increasing concentrations of STING agonist. 0.1 0.5 1 510 mg/mL mg/mL mg/mL mg/mL mg/mL 3′,5′-c-di-GMP 0.99 0.98 0.97 1.02 1.10ML-RS-CDA* 0.99 1.05 1.42 17.03 20.05 ML-RR-CDA** 1.02 1.09 1.17 9.0616.89 Example 1a 1.02 1.04 1.07 2.77 7.30 Example 1b 0.97 0.99 1.01 1.293.71 Example 2a 1.14 5.73 11.47 12.81 11.52 Example 2b 1.09 1.70 4.2613.22 13.68 Example 3a 0.99 0.96 0.99 1.01 1.04 Example 3b 0.98 0.991.00 1.08 1.14 Example 4d 0.21 nd 0.24 nd 0.36 *=dithio-[R_(p),S_(p)]-cyclic-[A(2′,5′)p-A(3′,5′)p]; **=dithio-[R_(p),R_(p)]-cyclic-[A(2′,5′)p-A(3′,5′)p]; prep'd as in WO2014/189805. Examples 4a-4c did not show significant activity.Mouse Ductal Pancreatic Cancer Assay

The procedure of Boj et al. (Cell 2015, 160, 324-338) was followed,which employs murine organoids. 2.5×10⁵ MT4-2D cells were injectedsubcutaneously on the right flank of male 6 week old C57BL/6J mice. 5 ugof the indicated STING agonist was injected intra-tumorally on day 15 ina volume of 50 ul.

TABLE 3 In Vivo Activity Example 2b Days Post ML-RR-CDA Example 2a(Tumor Vol, Challenge (Tumor Vol, mm³) (Tumor Vol, mm³) mm³) 0 0.0 0.00.0 4 19.0 29.2 28.4 6 56.3 64.3 53.9 8 81.9 84.3 66.5 10 84.5 103.387.4 12 107.3 125.9 98.3 14 110.9 113.8 106.6 15 125.6 132.1 114.7 16193.2 134.0 130.8 17 131.1 122.7 128.3 18 117.0 82.8 90.4 20 141.9 72.8115.3 22 147.2 91.6 64.9 25 154.0 124.0 70.4 27 241.4 142.0 80.7 29198.5 163.1 99.6 32 250.1 217.7 102.6

All references, patents or applications, U.S. or foreign, cited in theapplication are hereby incorporated by reference as if written herein intheir entireties. Where any inconsistencies arise, material literallydisclosed herein controls.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A compound of structural Formula I:

or a salt, ester, tautomer, or prodrug thereof, wherein: A₁ is CH and A₂is N, or A₁ is N and A₂ is CH, or A₁ is CH and A₂ is CH; R_(1a) andR_(1b) are independently selected from H and NH₂; R₂ is selected fromOH, F, Cl, N₃, and NH₂; R₃ is selected from OH, F, Cl, N₃, and NH₂;R_(4a) and R_(4b) are independently selected from NH₂, OH, NHR₅, andOR₅; and R₅ is independently selected from methyl, ethyl, and propyl,with the provisos that: when A₁ is CH, then R_(4a) is not NHR₅ or OR₅;and when A₂ is CH, then R_(4b) is not NHR₅ or OR₅.
 2. The compound asrecited in claim 1, wherein A₁ is CH and A₂ is N.
 3. The compound asrecited in claim 1, wherein A₁ is N and A₂ is CH.
 4. The compound asrecited in claim 1, wherein A₁ is CH and A₂ is CH.
 5. The compound asrecited in claim 1, wherein R_(4a) and R_(4b) are both NH₂.
 6. Thecompound as recited in claim 5, wherein R₂ is selected from F and Cl. 7.The compound as recited in claim 1 wherein the compound has structuralFormula II:

or a salt, ester, tautomer, or prodrug thereof, wherein: A₁ is CH and A₂is N, or A₁ is N and A₂ is CH, or A₁ is CH and A₂ is CH; R_(1a) andR_(1b) are independently selected from H and NH₂; R₂ is selected fromOH, F, and Cl; R₃ is OH; R_(4a) and R_(4b) are independently selectedfrom NH₂ and OH.
 8. The compound as recited in claim 7, wherein A₁ is CHand A₂ is N.
 9. The compound as recited in claim 7, wherein A₁ is N andA₂ is CH.
 10. The compound as recited in claim 7, wherein A₁ is CH andA₂ is CH.
 11. The compound as recited in claim 7, wherein R_(4a) andR_(4b) are both NH₂.
 12. The compound as recited in claim 11, wherein R₂is selected from F and Cl.
 13. The compound as recited in claim 1,wherein the compound is selected from

or a salt, ester, tautomer, or prodrug of either of the above compounds.14. The compound as recited in claim 1, wherein the compound is selectedfrom

or a salt, ester, tautomer, or prodrug of either of the above compounds.15. The compound as recited in claim 1, wherein the compound is selectedfrom

or a salt, ester, tautomer, or prodrug of any of the above compounds.16. The compound as recited in claim 1, wherein the compound is selectedfrom

or a salt, ester, tautomer, or prodrug of either of the above compounds.17. The compound as recited in claim 1, wherein the compound is selectedfrom

or a salt, ester, tautomer, or prodrug of any of the above compounds.18. A drug delivery vehicle comprising a compound of Formula I havingthe structure shown below or a salt, ester, tautomer, or prodrug thereofwhich is conjugated to a targeting moiety,

wherein: A₁ and A₂ are independently selected from CH and N; R_(1a) andR_(1b) are independently selected from H and NH₂; R₂ is selected fromOH, F, Cl, N₃, and NH₂; R₃ is selected from OH, F, Cl, N₃, and NH₂;R_(4a) and R_(4b) are independently selected from NH₂, OH, NHR₅, andOR₅; R₅ is independently selected from methyl, ethyl, and propyl; withthe proviso that: when A₁ is CH, then R_(4a) is not NHR₅ or OR₅; when A₂is CH, then R_(4b) is not NHR₅ or OR₅; and when A₁ and A₂ are both Nthen at least one of R₂ and R₃ is not OH.
 19. A drug delivery vehiclecomprising a compound as recited in claim 1 contained within a containermoiety.
 20. A pharmaceutical composition comprising a compound asrecited in claim 1 together with a pharmaceutically acceptable carrier.21. A method of agonism of STING comprising contacting STING with acompound as recited in claim
 1. 22. A method of treatment of aSTING-mediated disease comprising the administration of atherapeutically effective amount of a compound as recited in claim 1 toa patient in need thereof.
 23. A method for achieving an effect in apatient comprising the administration of a therapeutically effectiveamount of a compound as recited in claim 1 to a patient, wherein saideffect is chosen from induction of transcription of host defense genes,production of a cytokine, release of chemokines, priming ofantigen-specific T-cells.
 24. A method of inducing STING-dependent typeI interferon production in an individual comprising the administrationof a sufficient amount of a compound as recited in claim 1 to a patient.25. A method for administration of a vaccine to a subject comprising:(a) administration of an effective amount of said vaccine; and (b)administration of an effective amount of a compound as recited in claim1; wherein the efficacy of said vaccine is greater with administrationof said compound as recited in claim
 1. 26. The compound as recited inclaim 1, wherein R_(1a) and R_(1b) are both H.
 27. The compound asrecited in claim 26, wherein R_(4a) and R_(4b) are independentlyselected from NH₂ and NHR₅.
 28. The compound as recited in claim 27,wherein R_(4a) and R_(4b) are both NH₂.